Tumor-associated Macrophages In Tumors Research Articles

Blood vessels expressing CD90 in human and rat brain tumors.

Blood vessels in brain tumors, particularly glioblastomas, have been shown to express CD90. CD90(+) cells in and around blood vessels in cancers including brain tumors have been identified as endothelial cells, cancer stem cells, fibroblasts or pericytes. In this study, we aimed to determine the nature or type(s) of cells that express CD90 in human brain tumors as well as an experimental rat glioma model by double immunofluorescence staining. The majority of CD90(+) cells in human glioblastoma tissue expressed CD31, CD34 and von Willebrand factor, suggesting that they were endothelial cells. Vasculatures in a metastatic brain tumor and meningioma also expressed CD90. CD90(+) cells often formed glomeruloid structures, typical of angiogenesis in malignant tumors, not only in glioblastoma but also in metastatic tumors. Some cells in the middle and outer layers of the vasculatures expressed CD90. Similar results were obtained in the rat glioma model. There were cells expressing both α-smooth muscle actin and CD90 in the middle layer of blood vessels, indicating that smooth muscle cells and/or pericytes may express CD90. CD90(+) vasculatures were surrounded by tumor-associated macrophages (TAMs). Thus, in addition to endothelial cells, some other types of cells, such as smooth muscle cells, pericytes and fibroblasts constituting the vasculature walls in brain tumors expressed CD90. Because CD90 has been shown to interact with integrins expressed by circulating monocytes, CD90 might be involved in angiogenesis through recruitment and functional regulation of TAMs in tumors. CD90(+) vasculatures may also interact with tumor cells through interactions with integrins. Because CD90 was not expressed by vasculatures in normal brain tissue, it might be a possible therapeutic target to suppress angiogenesis and tumor growth.

Abstract 474: Regulation of epithelial plasticity of cancer cells by tumor-associated macrophages

Abstract Epithelial plasticity of cancer cells is considered to be critical for the metastatic process of cancer cells. Epithelial-mesenchymal transition (EMT) has been proved to associate with cancer metastatic dissemination from primary tumor site to distal metastatic organ. In contrast, accumulated evidence showed that the reversal process of EMT, i.e., mesenchymal-epithelial transition (MET), is critical for metastatic colonization. However, the mechanism for controlling the epithelial plasticity during metastatic process is unclear. Tumor-associated macrophages (TAMs) are the most abundant host immune cells in tumor microenvironments. TAMs have been shown to modulate cancer progression through regulating cancer cell growth and metastasis, and remodeling extracellular matrix. However, it is still unclear about the roles of TAMs in regulating epithelial plasticity during cancer metastasis. Here, we demonstrate the distinct characteristics of that the TAMs in primary and metastatic tumors. The TAMs in metastatic tumors carry a M2-like phenotype; in contrast, the TAMs in primary tumors are composed of heterogeneous population containing both M1-like and M2-like cells. Furthermore, we polarize human monocytes into M1-like and M2-like macrophages and treat cancer cells with M1 and M2 macrophages conditioned medium. We find that different macrophage subtypes have a distinct effect in modulating epithelial plasticity of cancer cells. Interestingly, polarized macrophages regulate cancer cell morphological changes through epigenetic regulation. Therefore, we propose that TAMs in tumor microenvironment may polarize into different subtypes and facilitate cancer metastatic dissemination. These findings have implications for designing alternative strategies aimed at preventing and inhibiting cancer metastasis. Citation Format: CHIH-CHAN LEE, Muh-Hwa Yang. Regulation of epithelial plasticity of cancer cells by tumor-associated macrophages. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 474. doi:10.1158/1538-7445.AM2015-474

Adipose tissue macrophage polarization by intermittent hypoxia in a mouse model of OSA: Effect of tumor microenvironment

Intermittent hypoxia (IH)-induces alterations in tumor-associated macrophages (TAMs) that are associated with adverse cancer outcomes, as reported in patients suffering from sleep apnea. Adipose tissues (AT) and bone-marrow (BM)-derived cells are the inferred sources of macrophages infiltrating malignant tumors. Here, the sources of TAMs and the phenotypic changes induced by IH in the ipsilateral and contralateral AT were investigated by using a syngeneic murine solid tumor model (TC1).C57/B6 male mice were exposed to either IH or room air (RA) for 6 weeks, with TC1 cells being inoculated in the 2nd week. Macrophage content, phenotype and tissue origin were assessed in tumors, and ipsilateral and contralateral AT.IH induced a ~2.2-fold increase in TAM tumor infiltration. However, differential responses in the tumor ipsilateral and contralateral AT emerged: IH increased infiltration of preferentially M1 macrophages in contralateral AT, while reductions in macrophages emerged in ipsilateral AT and primarily consisted of the M2 phenotype. These changes were accompanied by reciprocal increases in resident and BM-derived TAMs in the tumor.IH-induced phenotypic alterations in AT macrophages surrounding the tumor and their increased infiltration within the tumor may contribute to the accelerated tumor progression associated with IH.

Abstract LB-168: Perivascular, CXCR4-expressing macrophages in tumors promote relapse following chemotherapy

Abstract The relapse of cancer after frontline therapies like chemotherapy is a major clinical problem as tumors often grow back at the site of surgical removal and/or as metastases. Tumor-associated macrophages (TAMs) are a major cellular component of both primary and metastatic tumors, and promote such key processes in tumor progression as angiogenesis, immunosuppression, invasion, and metastasis. Gene expression profiling and immunostaining studies have identified both classically (M1) and alternatively (M2) activated TAMs in human and mouse tumors. Heavily M2-skewed TAMs - those expressing the angiopoietin receptor, TIE2 - have been shown to be important for angiogenesis and immunosuppression in mouse tumors. Moreover, high levels of TAMs expressing another M2 marker, CD163, correlate with poor prognosis in various forms of human cancer. TAMs have been shown recently to reduce the sensitivity of mouse mammary tumors to cytotoxic agents like paclitaxel (PTX) and doxorubicin. Here, we report on our use of various mouse tumor models to investigate the role of TAMs in tumor relapse after chemotherapy. Our studies show that a distinct subset of M2-skewed, CXCR4hi VEGF+ TAMs cluster around blood vessels after administration of various cytotoxic agents. This correlated with increased tumor CXCL12 levels - and the selective depletion of this perivascular TAM subset, using the CXCR4 antagonist, AMD3100 (Plerixafor), revealed an important role for these cells in driving revascularisation and regrowth in post-chemotherapy tumors. Additionally, adoptive transfer of this TAM subset into tumor-bearing mice after chemotherapy accelerated tumor regrowth. No such effect was seen with non-M2 skewed TAMs isolated from the same tumors. These studies suggest that combining chemotherapy with inhibitors of the CXCL12-CXCR4 axis could delay tumor relapse in cancer patients. Citation Format: Claire E. Lewis. Perivascular, CXCR4-expressing macrophages in tumors promote relapse following chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-168. doi:10.1158/1538-7445.AM2014-LB-168

Abstract 4797: VS-6063 (defactinib) targets cancer stem cells directly and through inhibition of tumor-associated macrophages and cytokine production

Abstract Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase which mediates integrin and growth factor receptor signaling to regulate tumor growth, metastasis and angiogenesis. FAK is also required for the proliferation/survival of cancer stem cells (CSCs). Protein tyrosine kinase 2 (PYK2) is the most closely related protein kinase to FAK, sharing 60% homology in the kinase domain and a similar role in cellular signaling pathways. Several lines of evidence suggest that dual targeting of FAK and PYK2 should confer greater antitumor efficacy than an agent which only inhibits FAK. Inhibition of mammary stem cells or tumor angiogenesis by FAK inhibition can be bypassed by PYK2 activity, suggesting advantages of dual FAK/PYK2 inhibition. Results of PYK2 knockout in mice suggest that PYK2 inhibition is likely to be well tolerated. Based on the observation of reduced macrophage infiltration in PYK2 knockout mice, we investigated effects of PYK2 inhibition on tumor-associated macrophages (TAMs). Abundance of TAMs has been correlated with poor prognosis in multiple cancer types including mesothelioma and breast cancer. VS-6063 is a potent and orally active inhibitor of FAK kinase activity. Preliminary clinical activity has been observed in a Phase 1 single agent trial and in an ongoing Phase 1/1b trial in combination with paclitaxel in patients with ovarian cancer. We find that in addition to potent FAK inhibition, VS-6063 also inhibits PYK2 kinase activity with nanomolar potency in both biochemical enzymatic and cellular autophosphorylation assays. Using both pharmacological FAK inhibitors and FAK siRNA, we have previously demonstrated that FAK inhibition effectively reduces CSCs. We report here that dual targeting of FAK and PYK2 by VS-6063 may more effectively suppress CSCs than inhibition of FAK alone. We find that human monocyte-derived macrophages release IL-6 and IL-8, and these cytokines increase the proportion of ALDH-positive CSCs in mesothelioma and breast cancer cell lines. VS-6063 dose-dependently inhibited production of IL-6 and IL-8, whereas a FAK-only reference inhibitor had no effect on these cytokines. Accordingly, PYK2 siRNA decreased IL-6 release from human macrophages, while FAK siRNA did not. In the MDA-MB-231 breast cancer xenograft model in vivo, VS-6063 reduced serum IL-6 while the FAK-only reference compound did not. Furthermore, VS-6063 substantially reduced the number of F4/80-positive macrophages in the tumors. These data support a model in which TAMs release IL-6 and IL-8 in a PYK2-dependent manner. Dual inhibition of FAK and PYK2 by VS-6063 effectively decreases both the presence of TAMs in tumors and the ability of TAMs to release cytokines that stimulate CSC proliferation and survival, and may therefore provide the opportunity for more durable clinical response than inhibition of FAK alone. Citation Format: Jonathan A. Pachter, Jennifer E. Ring, Vihren N. Kolev, Irina M. Shapiro, Christian M. Vidal, Mahesh V. Padval, Qunli Xu. VS-6063 (defactinib) targets cancer stem cells directly and through inhibition of tumor-associated macrophages and cytokine production. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4797. doi:10.1158/1538-7445.AM2014-4797

Correlation of tumor-associated macrophages with clinicopathologic factors and angiogenesis in gastric cancer.

54 Background: Tumor associated macrophages (TAMs) are major components of tumor environment, and polarized into M1 and M2 type. M1 has been known as antitumorigenic, whereas M2 as protumorigenic. M2 have a significant role in tumor progression by promoting tumor cell invasion, migration and angiogenesis. We evaluated the M2 macrophages to investigate its importance in predicting clinical outcome or prognosis, and the relationship between M2 and angiogenesis in patients with gastric cancer. Methods: Formalin-fixed, paraffin-embedded blocks were obtained from the 88 patients with gastric cancer. CD163+ TAMs and CD105+ vessels were evaluated by immunohistochemical staining and the extent of CD163+ TAMs in tumor were divided into three groups: (A) infiltrated TAMs in cancer cell nest (nest TAMs); (B) infiltrated TAMs in tumor stroma (stroma TAMs); and (C) infiltrated TAMs along the invasive margin of a tumor (margin TAMs). Results: The increased stroma and margin TAMs (>72 and >102, respectively) were closely correlated with lymph node metastasis, TNM stage and lymphatic invasion (p<0.05 in 3 factors) and positive correlation existed between the stroma and margin TAMs (p<0.001). Disease-free survival rate analyzed using the Kaplan– Meier method was significantly lower in patient with high stroma and margin TMAs than patient with low stroma and margin TAMs (p=0.0039 and 0.0499, respectively). CD105+ vessels showed significant correlation with lymph node metastasis and lymphatic invasion.(p=0.006 and p<0.0001, respectively) Stromal, marginal and nest TAMs were positively correlated with CD105+ vessels (p=0.001). Conclusions: M2 infiltration in tumor stroma and tumor margin in gastric cancer showed relationship with aggressive phenotypes such as lymph node metastasis, TNM stage and lymphatic invasion and was correlated with angiogenesis. This study supports the view that the adverse prognostic impact of M2 involves tumor angiogenesis in gastric cancer.

Abstract B283: Defactinib (VS-6063) targets cancer stem cells directly and through inhibition of tumor-associated macrophages and cytokine production.

Abstract Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase which mediates integrin and growth factor receptor signaling to regulate tumor growth, metastasis and angiogenesis. FAK is also required for the proliferation/survival of cancer stem cells (CSCs). Protein tyrosine kinase 2 (PYK2) is the most closely related protein kinase to FAK, sharing 60% amino acid identity in the kinase domain and a similar role in cellular signaling pathways. Several lines of evidence suggest that dual targeting of FAK and PYK2 should confer greater antitumor efficacy than an agent which only inhibits FAK. Inhibition of tumor angiogenesis or mammary stem cells by FAK inhibition can be bypassed by PYK2 activity, suggesting advantages of dual FAK/PYK2 inhibition. Accordingly, a critical role of PYK2 in CSC regulation and NSCLC patients’ prognosis has recently been reported (Kuang et al. 2013, Br J Cancer). Furthermore, preclinical data suggest that PYK2 inhibition can prevent growth of metastatic tumor cells in bone. Results of PYK2 knockout in mice suggest that PYK2 inhibition is likely to be well tolerated. Based on the observation of reduced macrophage infiltration in PYK2 knockout mice, we investigated effects of PYK2 inhibition on tumor-associated macrophages (TAMs). Abundance of TAMs has been correlated with poor prognosis in multiple cancer types including mesothelioma and breast cancer. Defactinib is a potent and orally active inhibitor of FAK kinase activity. Preliminary clinical activity has been observed in a Phase 1 single agent trial and in an ongoing trial in combination with paclitaxel in patients with ovarian cancer. We find that in addition to potent FAK inhibition, defactinib also inhibits PYK2 kinase activity with nanomolar potency in both biochemical enzymatic and cellular autophosphorylation assays. Using both pharmacological FAK inhibitors and FAK siRNA, we have previously demonstrated that FAK inhibition effectively reduces CSCs. We report here that dual targeting of FAK and PYK2 by defactinib may more effectively suppress CSCs than inhibition of FAK alone. We find that differentiated THP-1 macrophages release IL-6 and IL-8, and these cytokines increase the proportion of ALDH-positive CSCs in mesothelioma and breast cancer cell lines. Defactinib dose-dependently inhibited production of IL-6 and IL-8, whereas a FAK-only reference inhibitor had no effect on these cytokines. Furthermore, in MM87 mesothelioma and MDA-MB-231 breast cancer xenograft models, defactinib substantially reduced the number of F4/80-positive TAMs in the tumors. These data support a model in which TAMs release IL-6 and IL-8 in a PYK2-dependent manner. Dual inhibition of FAK and PYK2 by defactinib effectively decreases both the presence of TAMs in tumors and the ability of TAMs to release cytokines that stimulate CSC proliferation and survival, and may therefore provide the opportunity for more durable clinical response than inhibition of FAK alone. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B283. Citation Format: Jennifer E. Ring, Vihren N. Kolev, Irina M. Shapiro, Quentin G. Wright, Joseph R. Testa, Mahesh V. Padval, Qunli Xu, Jonathan A. Pachter. Defactinib (VS-6063) targets cancer stem cells directly and through inhibition of tumor-associated macrophages and cytokine production. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B283.

Phosphatidylserine-targeting antibody induces M1 macrophage polarization and promotes myeloid-derived suppressor cell differentiation.

Multiple tumor-derived factors are responsible for the accumulation and expansion of immune-suppressing myeloid-derived suppressor cells (MDSC) and M2-like tumor-associated macrophages (TAM) in tumors. Here, we show that treatment of tumor-bearing mice with docetaxel in combination with the phosphatidylserine-targeting antibody 2aG4 potently suppressed the growth and progression of prostate tumors, depleted M2-like TAMs, and MDSCs, and increased the presence of M1-like TAMs and mature dendritic cells in the tumors. In addition, the antibody markedly altered the cytokine balance in the tumor microenvironment from immunosuppressive to immunostimulatory. In vitro studies confirmed that 2aG4 repolarized TAMs from an M2- to an M1-like phenotype and drove the differentiation of MDSCs into M1-like TAMs and functional dendritic cells. These data suggest that phosphatidylserine is responsible for the expansion of MDSCs and M2-like TAMs in tumors, and that bavituximab, a phosphatidylserine-targeting antibody currently in clinical trials for cancer, could reverse this process and reactivate antitumor immunity.

Selective ablation of tumor‐associated macrophages suppresses metastasis and angiogenesis

Tumor-associated macrophages (TAM) play a critical role in promoting tumor development and metastasis. In the present study, we found that legumain, an asparaginyl endopeptidase, was highly expressed on the surface of TAM. A doxorubicin-based prodrug specifically activated by legumain selectively ablated TAM and resulted in a significant reduction of angiogenic factors and related tumor vessel growth. Treatment with the prodrug also suppressed circulating tumor cells and myeloid immune suppressor Gr-1+/CD11b+ cells in tumor-bearing animals. After selective ablation of TAM using the prodrug, tumor growth and metastases were greatly inhibited in murine tumor models. These results indicate that legumain-activated prodrugs targeting TAM in tumors might represent a novel anticancer strategy.

Abstract LB-339: Increase in tumor-associated macrophages (TAMs) after antiangiogenic therapy is associated with poor survival in recurrent glioblastoma (GBM) patients.

Abstract Antiangiogenic therapy is associated with increased radiographic responses in glioblastomas (GBMs), but tumors invariably recur. Because tumor-associated macrophages (TAMs) have been shown to mediate escape from antiangiogenic therapy in preclinical models, we examined the role of TAMs in recurrent glioblastoma (rGBM) patients. We compared autopsy brain specimens from 20 rGBM patients who received antiangiogenic treatment and chemoradiation (AAT+) to 8 patients who received chemotherapy and/or radiotherapy without antiangiogenic therapy, or no treatment (AAT-). TAMs were morphologically and phenotypically analyzed using flow cytometry and immunohistochemistry (IHC) for CD68, CD14, CD163, and CD11b expression. Flow cytometry showed an increase in TAMs in the AAT+ patients. IHC analysis demonstrated an increase in CD68+ TAMs in the tumor bulk (p<0.01) and infiltrative areas (p=0.02) in AAT+ patients. We also observed an increase in CD11b+ cells in the tumor bulk (p<0.01) and an increase in CD163+ TAMs in infiltrative tumor (p=0.02). Of note, an increased number of CD11b+ cells in bulk and infiltrative tumor (p=0.05 and p=0.05, respectively) correlated with poor overall survival in patients who first received antiangiogenic therapy at recurrence. In summary, rGBMs showed an increased infiltration in myeloid populations in the tumor bulk and in the infiltrative regions after antiangiogenic therapy. Higher numbers of CD11b+ cells correlated with poor survival in rGBM patients. These data suggest that TAMs may participate in escape from antiangiogenic therapy and may represent a potential biomarker of resistance and a potential therapeutic target in rGBM. Citation Format: Christine Lu-Emerson, Matija Snuderl, Nathaniel D. Kirkpatrick, Jermaine Goveia, Jennie Taylor, Christian Davidson, Yuhui Huang, Lars Riedemann, S. Percy Ivy, G. Dan Duda, Marek Ancukiewicz3, Scott R. Plotkin, Andrew Chi, Elizabeth R. Gerstner, April F. Eichler, Jorg Dietrich, Anat O. Stemmer-Rachamimov, Tracy T. Batchelor, Rakesh K. Jain. Increase in tumor-associated macrophages (TAMs) after antiangiogenic therapy is associated with poor survival in recurrent glioblastoma (GBM) patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-339. doi:10.1158/1538-7445.AM2013-LB-339

Abstract 3651: Phosphatidylserine-targeting antibody induces differentiation of myeloid-derived suppressor cells into M1-like macrophages

Abstract Introduction: The phosphatidylserine (PS)-targeting antibody, bavituximab, is currently in Phase IIb clinical trials in patients with lung cancer. Bavituximab, and its murine counterpart, 2aG4, induce the attack of monocytes and macrophages on PS-expressing tumor vascular endothelium and tumor cells and inhibit the immunosuppressive effects of PS in the tumor microenvironment. Myeloid-derived suppressor cells (MDSC) are one of the major cells responsible for the immunosuppressed state in tumors. In this study, we tested the influence of 2aG4 on the differentiation of MDSC into M1-like tumor associated macrophages (TAMs). Methods: MDSC were isolated from 4T1 tumor bearing mice with anti-Gr1-coated magnetic beads. The purified MDSC were then cultured for 5 days in the presence of 2aG4 or control C44 antibody. For in vivo studies, PC3 tumor-bearing mice were treated with 2aG4 for 30 days and MDSC and TAMs in tumors and spleens were analyzed by FACS and immunohistochemistry. Results: 2aG4 treatment of purified MDSC switched their cytokine production from an immunosuppressive IL-10-dominated response to a pro-inflammatory IL-12- and TNFα -dominated response. The percentage of Gr1+ cells decreased to 8% in the 2aG4-treated cultures (P<0.0001) but only to 50 – 57% in the PBS and C44-treated cultures. Treatment with 2aG4 induced the differentiation of MDSC into M1-like macrophages that expressed lower CD206 and produced more NO than control cultures. Treatment of mice bearing PC3 prostate tumors with 2aG4 decreased the percentage of MDSC from 7% to 4% (P<0.001) in the tumors and from 28% to 20% (P<0.001) in the spleens. The antibody treatment also increased the ratio of M1 to M2 TAMs in PC3 tumors from 0.7% to 1.4% (P<0.001). Conclusion: Taken together, our results suggest that 2aG4 causes the differentiation of MDSCs into macrophages having an M1-like phenotype. 2aG4 treatment decreased IL-10 production and increased IL-12 and TNFα-production. These results suggest that 2aG4 treatment reactivates innate immunity in tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3651. doi:10.1158/1538-7445.AM2011-3651

Impact of Tumor-Associated Macrophages in LHBETATAGMice on Retinal Tumor Progression: Relation to Macrophage Subtype

To determine the distribution of tumor-associated macrophages (TAMs) during retinoblastoma tumor development, examine the contribution of bone marrow-derived TAMs in retinoblastoma tumors, and evaluate the supportive role of TAMs in tumor growth in a transgenic retinoblastoma mouse model. The time course of macrophage infiltration in transgenic retinoblastoma tumors was assessed by immunohistochemistry at different time points in tumorigenesis. The origin of TAMs in transgenic retinoblastoma tumors was determined by transplanting 10(7) bone marrow cells from green fluorescent protein (GFP)-positive 16-week-old mice into age-matched, irradiated LH(BETA)T(AG) mice via tail vein injections. Macrophage depletion was performed by subconjunctival (SC) delivery of liposomal clodronate. The density of TAMs increased from 4 to 12 weeks of age in mice with small to medium tumors (P = 0.037) and remained stable in the later stages of disease (i.e., 16 weeks old with large tumors; P = 0.20). In 16-week-old mice, 38% (2.5 +/- 3.2 cells per 400x high-power field) of TAMs were GFP-positive, bone marrow-derived macrophages. Total TAM depletion was associated with a significant decrease in the expression levels of MMP-9 (P = 0.014) and mature vessels (P < 0.001) and a nonsignificant decrease in the density of neovessels (P = 0.94). The density of M2-polarized TAMs did not change significantly after TAM depletion (P = 0.68). After M1-polarized TAM depletion, the tumor burden increased (P = 0.056). This work extends understanding of the complex role that macrophages play in retinoblastoma. Macrophage modulation in the tumor microenvironment is a critical factor in retinoblastoma tumor progression.

Investigation into the Role of Tumor-Associated Macrophages in the Antitumor Activity of Doxil

PurposeOur recent studies show specific localization of long-circulating liposomes (LCL) within the endosomal/lysosomal compartment of tumor-associated macrophages (TAM). Based on this finding, the present study aims to investigate whether clinically applied LCL formulations such as Doxil (LCL-encapsulated doxorubicin), have alternative mechanisms of action additionally to direct drug-mediated cytotoxicity towards tumor cells.MethodsThe antitumor activity of Doxil was evaluated in B16.F10 melanoma-bearing mice, in the presence and in the absence of TAM. To suppress TAM functions, liposomal clodronate (Lip-CLOD) was injected 24 h before the actual treatment. The effect of Doxil on the levels of angiogenic factors was determined using an angiogenic protein array. As positive control, the same experiments were conducted with LCL-encapsulated prednisolone phosphate (LCL-PLP), a tumor-targeted formulation with known strong anti-angiogenic/anti-inflammatory effects on TAM.ResultsOur results show that the antitumor efficacy of Doxil was only partially attributed to the inhibition of TAM-mediated angiogenesis whereas LCL-PLP inhibited tumor growth through strong suppressive effects on pro-angiogenic functions of TAM. As described previously, the main mechanism of Doxil might be a cytotoxic effect on tumor cells.ConclusionsOur findings suggest that the antitumor activity of Doxil does not depend mainly on the presence of functional TAM in tumors.