tumor associated macrophages (TAM)

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yoa
yoa's picture
tumor associated macrophages (TAM)

Hello,

Please does anyone know how I can isolate and culture tumor associated macrophages cells

Thank you in advance

Tony Rook
Tony Rook's picture
Hello yoa:

Hello yoa:

Here are some reference links that may help you isolate macrophages cells associated with tumors.

Kumagai K, Itoh K, Hinuma S, Tada M. Pretreatment of plastic Petri dishes with fetal calf serum. A simple method for macrophage isolation. J Immunol Methods. 1979;29(1):17-25.
doi:10.1016/0022-1759(79)90121-2

Abstract:
We have developed a simple method which can recover the highly purified macrophages or monocytes in suspension from mouse peritoneal exudate cells and human perpheral blood mononuclear cells. Plastic Petri dishes coated overnight with heat-inactivated fetal calf serum (FCS) selectively bind macrophages and monocytes. The adherent macrophages and monocytes are easily removed by incubation in phosphate-buffered saline containing 0.2% ethylenediamine tetraacetate (EDTA) and 5% FCS, and recovered as a cell suspension with greater than 95% purity. A small number of isolated cells can restore the mitogenic response to phytohemagglutinin (PHA-P) of macrophages-depleted lymphocytes and can lyse 51Cr-labeled target cells in an antibody-dependent cell-mediated cytotoxicity system. Thus, the method should be valuable for studies of various functions of macrophages and monocytes from different immune tissues of man and animals.

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Sebastian F. Schoppmann, Peter Birner, Johannes Stckl, Romana Kalt, Robert Ullrich, Carola Caucig, Ernst Kriehuber, Katalin Nagy, Kari Alitalo and Dontscho Kerjaschki. Tumor-Associated Macrophages Express Lymphatic Endothelial Growth Factors and Are Related to Peritumoral Lymphangiogenesis. American Journal of Pathology. 2002;161:947-956.

Abstract:
Formation of lymphatic metastasis is the initial step of generalized spreading of tumor cells and predicts poor clinical prognosis. Lymphatic vessels generally arise within the peritumoral stroma, although the lymphangiopoietic vascular endothelial growth factors (VEGF)-C and -D are produced by tumor cells. In a carefully selected collection of human cervical cancers (stage pT1b1) we demonstrate by quantitative immunohistochemistry and in situ hybridization that density of lymphatic microvessels is significantly increased in peritumoral stroma, and that a subset of stromal cells express large amounts of VEGF-C and VEGF-D. The density of cells producing these vascular growth factors correlates with peritumoral inflammatory stroma reaction, lymphatic microvessel density, and indirectly with peritumoral carcinomatous lymphangiosis and frequency of lymph node metastasis. The VEGF-C- and VEGF-D-producing stroma cells were identified in situ as a subset of activated tumor-associated macrophages (TAMs) by expression of a panel of macrophage-specific markers, including CD68, CD23, and CD14. These TAMs also expressed the VEGF-C- and VEGF-D-specific tyrosine kinase receptor VEGFR-3. As TAMs are derived from monocytes in the circulation, a search in peripheral blood for candidate precursors of VEGFR-3-expressing TAMs revealed a subfraction of CD14-positive, VEGFR-3-expressing monocytes, that, however, failed to express VEGF-C and VEGF-D. Only after in vitro incubation with tumor necrosis factor-{alpha}, lipopolysaccharide, or VEGF-D did these monocytes start to synthesize VEGF-C de novo. In conclusion VEGF-C-expressing TAMs play a novel role in peritumoral lymphangiogenesis and subsequent dissemination in human cancer.

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Sergei Kusmartsev and Dmitry I. Gabrilovich. STAT1 Signaling Regulates Tumor-Associated Macrophage-Mediated T Cell Deletion. The Journal of Immunology, 2005, 174: 4880-4891.

Abstract:
It is well established that tumor progression is associated with the accumulation of myeloid suppressive cells, which in mice include Gr-1+ immature myeloid cells and F4/80+ macrophages. The paradox is that with the exception of terminal stages of the disease or chemotherapy treatment, tumor-bearing mice or cancer patients do not display a profound systemic immune suppression. We therefore raised the question as to whether myeloid cell-mediated T cell suppression is controlled at a local level at the site of the tumor. We have demonstrated that after adoptive transfer to tumor-bearing recipients, Gr-1+ (immature myeloid cells) freshly isolated from spleens of tumor-bearing mice become F4/80+ tumor-associated macrophages (TAM). These TAM, but not F4/80+ macrophages or Gr-1+ cells freshly isolated from spleens of tumor-bearing or naive mice were able to inhibit T cell-mediated immune response in vitro via induction of T cell apoptosis. Arginase and NO were both responsible for the apoptotic mechanism, and were seen only in TAM, but not in freshly isolated Gr1+ cells. Using the analysis of STAT activity in combination with STAT knockout mice, we have determined that STAT1, but not STAT3 or STAT6, was responsible for TAM-suppressive activity.

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Jiasen Cheng, De-Hua Huo, Dong-Ming Kuang, Jine Yang, Limin Zheng and Shi-Mei Zhuang. Human Macrophages Promote the Motility and Invasiveness of Osteopontin-Knockdown Tumor Cells. Cancer Research 67, 5141-5147, June 1, 2007.
doi: 10.1158/0008-5472.CAN-06-4763

Abstract:
Increasing evidence indicates that macrophages in tumor stroma can significantly modify the malignant phenotypes of tumors. Osteopontin (OPN) is frequently overexpressed in cancers with high metastatic capacity and, thus, has been considered as a potential therapeutic target. To find out whether macrophages can affect the outcome of OPN-knockdown tumor cells, we used RNA interference (RNAi) to stably silence the OPN expression in the highly invasive human hepatoma cell line SK-Hep-1. Silencing of OPN markedly decreased the motility and invasiveness of the SK-Hep-1 cells. Further studies using this cell model revealed that coculture with human macrophages or macrophage-conditioned medium largely restored the migration and invasion potential of OPN-knockdown tumor cells. Moreover, such macrophage-promoted motility can be effectively blocked either by the addition of OPN-neutralizing antibody to the cocultured medium or by silencing OPN expression in macrophages. These results indicate that macrophage-derived OPN can compensate for the decrease of OPN and thereby restore the metastatic potential of OPN-knockdown tumor cells. Further characterization of the underlying mechanisms disclosed that macrophage-derived OPN exerted its function independently of the actin cytoskeleton rearrangement or the activation of matrix metalloproteinase and Rho families. Our results suggest that there are fine-tuned complex interactions between cancer cells and stroma cells, which may modify the outcome of cancer therapy, and therefore should be considered for the rational design of anticancer strategy.

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hrystelle Lamagna, Michel Aurrand-Lions and Beat A. Imhof. Dual role of macrophages in tumor growth and angiogenesis. Journal of Leukocyte Biology. 2006;80:705-713.
doi:10.1189/jlb.1105656

Abstract:
During the neoplastic progression, macrophages as well as dendritic and NK cells are attracted into the tumor site and initiate the immune response against transformed cells. They activate and present tumor antigens to T cells, which are then activated to kill tumor cells. However, tumor cells are often capable of escaping the immune machinery. As the immune surveillance is not sufficient anymore, tumor-associated macrophages contribute to tumor progression. It is notable that tumor-associated macrophages promote the proliferation of tumor cells directly by secreting growth factors. They also participate in tumor progression by acting on endothelial cells and thus promoting the neovascularization of the tumor. Tumor-associated macrophages are indeed key protagonists during angiogenesis and promote each step of the angiogenesis cascade.

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Claire E. Lewis and Jeffrey W. Pollard. Distinct Role of Macrophages in Different Tumor Microenvironments. Cancer Research 66, 605-612, January 15, 2006

Abstract:
Macrophages are prominent in the stromal compartment of virtually all types of malignancy. These highly versatile cells respond to the presence of stimuli in different parts of tumors with the release of a distinct repertoire of growth factors, cytokines, chemokines, and enzymes that regulate tumor growth, angiogenesis, invasion, and/or metastasis. The distinct microenvironments where tumor-associated macrophages (TAM) act include areas of invasion where TAMs promote cancer cell motility, stromal and perivascular areas where TAMs promote metastasis, and avascular and perinecrotic areas where hypoxic TAMs stimulate angiogenesis. This review will discuss the evidence for differential regulation of TAMs in these microenvironments and provide an overview of current attempts to target or use TAMs for therapeutic purposes.

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Dontscho Kerjaschki. The crucial role of macrophages in lymphangiogenesis. J. Clin. Invest. 115:2316-2319 (2005).
doi:10.1172/JCI26354.

Abstract:
Lymphangiogenesis is associated with pathological processes such as the metastatic spread of carcinoma cells and organization of immunologically active lymphocytic infiltrates following organ transplantation. It has not yet been established whether expansion of the lymphatic vascular meshwork is driven by incorporation of progenitor cells or by local endothelial cell division. In this issue of the JCI, Maruyama et al. provide evidence that after mouse corneal transplant, CD11b+ macrophages infiltrate the corneal stroma and transdifferentiate into lymphatic endothelial cell clusters that join existing lymphatic vessels. In complementary in vitro experiments, murine peritoneal macrophages expressed lymphatic endothelial markers and formed vessel-like protrusions. These findings add yet another facet to the plasticity of macrophages, which are already known to transform from naive monocytes into VEGF-Cproducing cells. Thus, macrophages support lymphangiogenesis in 2 different ways, either by transdifferentiating and directly incorporating into the endothelial layer or by stimulating division of preexistent local lymphatic endothelial cells.

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Hope these help lead you in the right direction...

Tony Rook
Tony Rook's picture
Here are some additional

Here are some additional references that I ran across which details some macrophage isolation & purification methods...

M. A. Schon-Hegrad and P. G. Holt. Improved method for the isolation of purified mouse peritoneal macrophages. Journal of Immunological Methods
Volume 43, Issue 2, 16 June 1981, Pages 169-173
doi:10.1016/0022-1759(81)90020-X

Abstract:
Mouse peritoneal macrophages were cultured for 45 min in medium supplemented with fetal calf serum (FCS) in petri dishes coated overnight with heat-inactivated FCS. After removal of non-adherent cells by washing, adherent cells were detached by a brief incubation in the presence of sub-toxic levels of ethylenediamine tetraacetate (EDTA). Overall peritoneal macrophage recoveries of 90% can be routinely achieved with this method, and full cell viability is maintained.

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P. R. Woodb, R. J. Simesb, and D. S. Nelson. Activity of mouse macrophages purified by adherence to, and removal from, a plastic surface. Journal of Immunological Methods
Volume 28, Issues 1-2, 10 July 1979, Pages 117-124
doi:10.1016/0022-1759(79)90333-8

Abstract:
Mouse peritoneal exudate macrophages were allowed to adhere to plastic Petri dishes and, after washing, were removed by means of EDTA. Cells with the morphology of macrophages were recovered in a fair degree of purity (9098%) but in low yield (3134%). The macrophages recovered were fully active in the following ways: re-adherence to glass; pinocytosis of colloidal gold; phagocytosis of opsonized sheep erythrocytes; binding cytophilic antibody; production of lymphocyte activating factor; cytotoxic and cytostatic effects on tumour cells.

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Shinsuke Mikamo. A novel method for the purification of sheep red cell rosetting lymphocytes. Journal of Immunological Methods
Volume 107, Issue 2, 16 March 1988, Pages 189-196
doi:10.1016/0022-1759(88)90217-7

Abstract:
A rapid, direct method for the purification of sheep red cell rosetting lymphocytes (ERFC) was developed. The whole procedure, including rosette formation, density separation and hemolysis could be completed within 10 min. A mixture of human peripheral blood mononuclear cells (PBMC) and 2-aminoethylisothiouronium bromide hydrobromide-treated sheep erythrocytes (EAET) was layered onto Ficoll-Paque without any pretreatment and centrifuged at 600 × g for 2.5 min. The pellet was then immediately treated with an NH4Cl solution containing 10% FCS and hemolysis was completed within 1 min. The purity of ERFC separated in one cycle of the procedure was 98%, the viability 99% and the yield 56% of the initial lymphocyte count. The re-rosetting ability of the prepared cells, after hemolysis, was 95%. The lymphocytes in the fraction prepared by the same method contained 94.3% CD2(OKT11)+ cells, 90% of which were CD3(OKT3)+ cells (T cells) and 9% were CD16(Leulla)+ cells (NK cells).

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H. Lorraine Thompson, Joan Stevenson and J. H. Brock. The effect of iron and agar on production of hydrogen peroxide by stimulated and activated mouse peritoneal macrophages. FEBS Letters. Volume 200, Issue 2, 12 May 1986, Pages 283-286.
doi:10.1016/0014-5793(86)81153-X

Abstract:
The effect of iron on H2O2 production by mouse peritoneal macrophages exposed to opsonised zymosan has been investigated. Macrophages elicited with thioglycollate broth produced less H2O2 than macrophages activated by Corynebacterium parvum, and levels were not affected by prior incubation of the cells with 0.1 mM iron nitrilotriacetate. However, preincubation with the iron chelator desferrioxamine (1 mM) reduced H2O2 production by both types of macrophages. Incubation of macrophages with agar, a component of thioglycollate broth, also reduced H2O2 production, particularly by C. parvum-activated macrophages. The results indicate that although iron appears to be necessary for H2O2 production by macrophages, the low level of production by thioglycollate-elicited macrophages is not due to an inadequate level of metabolically utilisable iron, but may be a result of prior ingestion of agar present in the broth.

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Dina G. Fischer, William J. Hubbard and Hillel S. Koren. Tumor cell killing by freshly isolated peripheral blood monocytes. Cellular Immunology Volume 58, Issue 2, 1 March 1981, Pages 426-435
doi:10.1016/0008-8749(81)90235-5

Abstract:
Human peripheral blood monocytes were reproducibly shown to lyse a variety of tumor cells in a 3- to 4-hr 51Cr release assay. Ficoll-Hypaque-purified mononuclear cells were suspended in medium supplemented with either 10% autologous serum or fetal calf serum (PCS). With either serum, highly purified (9799%) and viable (>99%) monocyte suspensions were obtained by EDTA-reversible adherence to plastic surfaces which had been precoated with autologous serum. When used as effectors in cytotoxicity assays, the monocytes recovered from mononuclear cells suspended in FCS-supplemented medium exhibited higher cytolytic activity and were therefore used for further studies. Using FCS for both coating the plates and supplementing the suspension medium resulted in monocytes with low cytolytic activity. Tumor cell lysis measured by 51Cr release was detected within 2 hr of incubation and increased gradually with time. The level of lysis was dependent on the effector/target ratio and the tumor target cell employed. The involvement of natural killer lymphocytes in the observed tumoricidal activity was excluded. Detection of cytotoxic activity in a short-term assay will be very helpful in further studies of the mechanism of tumor cell killing by human monocytes since potential complicating effects of long-term in vitro cultivation will be minimized.

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S. Grace, L. A. Guthrie and R. B. Johnston, Jr. The use of mouse serum and the presence of non-adherent cells for the culture of mouse macrophages. Journal of Immunological Methods Volume 114, Issues 1-2, 10 November 1988, Pages 21-26.
doi:10.1016/0022-1759(88)90148-2

Abstract:
Mouse serum (MS) was investigated as an alternative to fetal calf serum (FCS) as a medium supplement for the culture of murine macrophages. Peritoneal macrophages were successfully cultured in medium supplemented with 120% MS and were able to produce superoxide anions in response to stimulation with phorbol myristate acetate (PMA) and to phagocytose antibody-coated erythrocytes effectively. Macrophages cultured in the presence of 5% or 20% FCS showed a generally augmented response to PMA, raising the possibility that they had been primed by constituents or contaminants of FCS. Lipopolysaccharide-elicited macrophages initially showed vigorous in vitro responses to PMA which decreased with increasing culture time in MS-supplemented medium. This de-differentiation of elicited macrophages could be due to the absence of LPS contamination and foreign protein when autologous serum is used as a medium supplement. In all cultures the presence of non-adherent cells for the first 24 h increased the number and superoxide response of adherent cells.

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Marie-Thérèse Chteau, Herisoa Rabesandratana and René Caravano. Suspended mouse peritoneal macrophages Preparation and properties. Journal of Immunological Methods. Volume 143, Issue 1, 20 September 1991, Pages 103-109.
doi:10.1016/0022-1759(91)90278-N

Abstract:
Since macrophages (MPH) are able to adhere firmly to solid surfaces, the recovery of viable and functional MPH has proven to be extremely difficult. We have developed a simple method using agarose coating for preparing MPH and culturing the cells in suspension. Their properties were tested over 72 h. The oxidative burst declined with time, but could be restored using the lymphokine rich supernatant of pokeweed-stimulated mouse spleen cells. In contrast, phagocytosis and Candida intra-cellular killing remained unchanged.

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Good luck!

Please let the board know which (if any) of these references helped isolate the best method for macrophage isolation / purification.

NANDACLONE92
NANDACLONE92's picture
 WE CAN ISOLATE THE MONOCYTES

 WE CAN ISOLATE THE MONOCYTES FROM BLOOD AND CONVERT INTO TAMS BY CHEMOKINES