This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Forssell, J. Articles by Palmqvist, R. Search for Related Content PubMed PubMed Citation Articles by Forssell, J. Articles by Palmqvist, R.

High Macrophage Infiltration along the Tumor Front Correlates with Improved Survival in Colon Cancer

Authors' Affiliations: ¹ Department of Medical Biosciences, Pathology, and ² Department of Surgical and Perioperative Sciences, Surgery, Umeå University, Umeå, Sweden; and ³ Pathologisches Institut der Ludwig-Maximilians Universität, Munich, Germany

Requests for reprints: Richard Palmqvist, Department of Medical Biosciences, Pathology, Umeå University, SE-901 85 Umeå, Sweden. Phone: 46-90-785-1532; Fax: 46-90-785-2829; E-mail: richard.palmqvist{at}medbio.umu.se.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Purpose: The role of macrophages in tumorigenesis is complex because they can both prevent and promote tumor development.

Experimental Design: Four hundred forty-six colorectal cancer specimens were stained with the pan-monocyte/macrophage marker CD68, and average infiltration along the tumor front was semiquantitatively evaluated using a four-grade scale. Each section was similarly scored for the presence of CD68 hotspots. Some aspects of macrophage-tumor cell interactions were also studied using in vitro coculture systems.

Results: Including all patients, regardless of surgical outcome and localization, survival increased incrementally with CD68TF_Mean infiltration grade (P = 0.0001) but not in curatively resected colon cancers (P = 0.28). CD68 hotspot score (CD68TF_Hotspot) was divided into high and low. A high hotspot score conferred a highly significant survival advantage also in curatively resected colon cancer cases (n = 199, P = 0.0002) but not in rectal cancers. CD68TF_Hotspot high turned out as an independent prognostic marker for colon cancer in multivariate analyses including gender, age, localization, grade, stage, tumor type, and lymphocytes at the tumor front, conferring a relative risk of 0.49 (P = 0.007). In vitro coculture experiments, using phorbol 12-myristate 13-acetate–activated U937 cells as macrophage model, revealed that a high ratio of macrophages to colon cancer cells inhibited cancer cell growth. This was partially dependent on cell-to-cell contact, whereas Boyden chamber cocultivation without cell-to-cell contact promoted cancer cell spread.

Conclusions: In conclusion, our data indicate that a dense macrophage infiltration at the tumor front positively influences prognosis in colon cancer and that the degree of cell-to-cell contact may influence the balance between protumorigenic and antitumorigenic properties of macrophages.

Given the somewhat variable results in different cancer types, we considered it an urgent task to clarify how macrophages affect prognosis in colorectal cancers. To address this issue, we investigated macrophage infiltration along the tumor front in 446 unselected CRC specimens and correlated the results to various clinicopathologic variables in univariate as well as multivariate analyses. In addition, we analyzed some aspects of macrophage influence on colon cancer cell behavior in vitro.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Clinical samples. We studied tumor specimens from 488 consecutive patients diagnosed with colorectal cancer and tissues collected during primary tumor surgical resection over the period 1995-2003 at Department of Surgery, Umeå University Hospital, Sweden. Ten specimens were excluded due to lack of adequate tissue available (i.e., tumor front included in the specimen, or inadequate staining results for CD68). An additional 32 patients lacked follow-up data or died from perioperative complications, leaving 446 patients for survival analyses. Of these, 82 patients received preoperative radiotherapy (5 x 5 Gy), 39 received long-term radiotherapy (25 x 2 Gy), and 113 received postoperative adjuvant chemotherapy (mainly 5-fluorouracil/leucovorin).

All routinely stained sections were reviewed by one observer (R.P.), who did all histopathologic classifications including stage, grade, tumor type (mucinous or nonmucinous), growth pattern (pushing or infiltrating), and lymphocytic reaction at the tumor front. Clinical data were obtained by reviewing the patient records (Å.Ö.) and survival data were collected during spring 2005. The study was approved by the local ethical committee of Umeå University.

Immunohistochemistry. Specimens were, according to routine procedures at the Department of Clinical Pathology, Umeå University Hospital, fixed in 4% formaldehyde and embedded in paraffin. One 4-µm section from each patient was cut, dried, dewaxed, and rehydrated before microwave treatment in citrate buffer (pH 6.0) for 3 x 5 min. A semiautomatic staining machine (Ventana ES, Ventana, Inc., Tucson, AZ) was used for the immunohistochemical procedures. Anti-CD68 monoclonal antibody (KP-1, DakoCytomation, Glostrup, Denmark) was used at a concentration of 1:4,000. The slides were counterstained with hematoxylin.

CD68 evaluation. CD68 immunostaining was evaluated along the tumor front over the whole section (7-10 view fields per section) and average infiltration (CD68TF_Mean) was semiquantitatively graded as no/weak (grade 1), moderate (grade 2), strong/robust (grade 3), and massive infiltration (grade 4). Tumors classified as 1 included totally negative specimens as well as specimens containing some scattered CD68-positive cells along the tumor margin. Tumors were classified as 2 when CD68 staining was continuous along the tumor margin but was not extended from the tumor front more than one cell layer on average. CD68 staining that, on average, extended two to three cell layers from the tumor margin over the whole section was classified as 3; whereas to be classified as 4, CD68 staining should extend several cell layers from the tumor margin in all fields. Examples of classified tumors are given in Fig. 1 . The specimens were evaluated twice by the same observer without any knowledge about prognosis or clinicopathologic variables. Eighty-seven percent of the specimens were judged identically between evaluations. Disagreements were evaluated a third time followed by a conclusive judgment. CD68 hotspots (CD68TF_Hotspot) were defined as infiltration grade of the two highest view fields, evaluated as for CD68TF_Mean, at a total magnification of x200 and graded from 1 to 4. Intraobserver agreements for CD68TF_Hotspot were 77%.

View larger version (56K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Examples of stainings representing the different grades of macrophage infiltration; no/low (A), moderate (B), high (C), and massive (D).

Cell cultures. HCT-116 and HT-29 cells were grown in 25-cm³ culture flasks in 7 mL DMEM/10% FCS. Media were changed every third day and cells were harvested with trypsin-EDTA (PBS containing 0.53 mmol/L EDTA, 145 mmol/L NaCl, and 0.05% trypsin). U937 cells were activated for 48 h with 20 ng/mL phorbol 12-myristate 13-acetate (PMA) to promote macrophage differentiation (30, 31) and carefully decanted to remove nonadherent cells. Adherent cells were harvested by two subsequent incubations in PBS containing 25 mmol/L EDTA (no trypsin) for 10 min. PMA-activated U937 cells were washed twice in DMEM/10% FCS and subsequently added in different amounts directly to the culture plate containing preadhered cancer cells. HCT-116 or HT-29 cells (0.30 x 10⁶) were seeded into a 25-cm² cell culture flask (1.2 x 10⁴/cm²) and allowed to adhere and grow for 24 h; after which, PMA-activated U937 cells were added. After 2 days, nonadherent cells were gently washed away with PBS prewarmed to 37°C; after which, adherent cells were harvested with trypsin-EDTA and viable count was determined by trypan blue exclusion. Cells were stained with antihuman CD45 to detect remaining U937 cells, and only CD45^– cells [65-95% of the recovered cells as determined by fluorescence-activated cell sorting (FACS)] were considered cancer cells.

For Boyden chamber cultures, 0.35 x 10⁵ HCT-116 or HT-29 cells were seeded in 1 mL DMEM/10% FCS into each well of a 24-well plate. After 24 h, medium was replaced with 0.5-mL prewarmed fresh medium and the appropriate amount of PMA-activated U937 cells (in 250 µL DMEM) was added either directly into the well or into the Boyden chamber insert (0.4-µm pores; Millipore Corp., Bedford, MA). After 2 days, the adherent cancer cells were gently rinsed with 37°C PBS and harvested with trypsin-EDTA, counted, and stained for CD45.

Macrophage-conditioned medium. U937 cells were cultured at 0.5 x 10⁶/mL in DMEM, 10% FCS containing 20 ng/mL PMA for 48 h. Nonadherent cells were discarded and remaining cells were washed twice in PBS prewarmed to 37°C and once in complete DMEM with 15-min incubation, and finally overlaid with DMEM containing 10% FCS (8 mL for a 75-cm³ flask). After incubation for 24 h, the medium was filtered and used as macrophage-conditioned medium.

Migration assay. HCT-116 cells (50,000) or HT-29 cells (100,000) were placed in a 24-well cell culture insert (8 µm pore size; BD Biosciences, Franklin Lakes, NJ) in 400-µL DMEM containing 2% FCS and allowed to adhere for 2 to 3 h. Next, culture inserts were placed in 500-µL DMEM containing 10% FCS (control) or in 500-µL macrophage-conditioned DMEM, 10% FCS and incubated for 20 h. After washes in PBS, inserts were placed in ice-cold methanol for 1 min and washed again in PBS. Cells adhering to the inside of the insert were thoroughly scraped of with a cotton top and the insert placed in a solution of 0.5% crystal violet for 10 min. After washes in PBS, the filter was cut out and cells were counted at 100x in three randomly selected fields using a 10x ocular with a grid pattern.

Immunofluorescence. HCT-116 and HT-29 cells were seeded in 24-well culture plates over a cover glass. Twenty-four hours later, cells were overlaid with a culture insert containing PMA-activated U937 cells and incubated for 48 h. After fixation in 4% paraformaldehyde, cells were stained with a rabbit ß-catenin antibody (Sigma, St. Louis, MO) and revealed with a Cy2-conjugated secondary goat anti-rabbit (Amersham, GE Healthcare, Little Chalfont, United Kingdom).

FACS. Cells were washed twice in FACS staining medium (PBS supplemented with 3% FCS) and incubated for 30 min on ice with FITC-conjugated antihuman CD45 (ImmunoTools GmbH, Friesoythe, Germany). Cells were washed twice, resuspended in FACS staining medium, and analyzed on a FACSCalibur (Becton Dickinson, Mountain View, CA).

	Results

Top Abstract Materials and Methods Results Discussion References

 
CD68 expression. The majority of CD68-positive cells were located in the stroma and, in particular, along the tumor front. CD68-positive cells were mostly in apparent direct contact with or immediately adjacent to tumor cells lining the tumor front. Examples of representative staining fields of respective grade are given in Fig. 1. Although the majority of tumors displayed a fairly homogeneous CD68 infiltration pattern along the tumor front, there were also tumors containing small areas that showed CD68 infiltration considerably above average grade. This prompted us to evaluate CD68TF_Hotspot, similarly graded from 1 to 4. The frequencies of CD68TF_Hotspot and CD68TF_Mean are shown in a cross-tabulation (Table 1 ).

View larger version (9K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Kaplan-Meier plots of all cancer specimens with follow-up data (n = 446) scored for CD68TFMean (– – –, 1; –––, 2; —, 3; –-–-–, 4) (A). Subgroup analysis of CD68TFHotspot for curatively resected colon cancers (n = 199; —, high; – – –, low; B) and for curatively resected rectal cancers (n = 125; —, high; – – –, low; C).

View larger version (11K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. PMA-activated U937 cells exert a cell-to-cell contact dependent growth inhibition on colon cancer cells in vitro. A, PMA-activated U937 cells were discriminated from tumor cells based on their expression of CD45. B, the number of viable cells recovered from wells with HCT-116 cells growing without U937 cells, overlaid with untreated U937 cells (5:1 macrophage to tumor cell ratio; hatched column), and overlaid with PMA-activated U937 cells (5:1 ratio; black column). The cell recovery is normalized to the control cells grown without macrophages (white column; set to 100%). Columns, mean calculated from two independent experiments; bars, SE. HCT-116 (C) or HT-29 (D) cells grown in 24-well plates without or with PMA-activated U937 cells in direct contact, or in Boyden chamber inserts, at 4:1 (hatched columns) and 10:1 (black columns) ratios. Columns, mean calculated from three independent experiments; bars, SE.

View larger version (36K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Macrophage-conditioned medium promotes enhanced migration of colon cancer cells. A, colon cancer cells that adhered to the inside of a culture insert were allowed to migrate toward control medium (left) or macrophage-conditioned medium (right) for 20 h. B, average of migrating cells calculated from three fields per experiment. Columns, mean of two independent experiments; bars, SE. C, distribution of E-cadherin in HT-29 cells untreated or treated for 48 h with macrophages communicating through a 0.4-µm culture insert. D, nuclear translocation of ß-catenin in HT-29 and HCT-116 cells treated for 48 h with macrophages communicating through a 0.4-µm culture insert.

	Discussion

Top Abstract Materials and Methods Results Discussion References

Our results were somewhat unexpected, given the dominating protumorigenic role in some other malignancies (2) and the fact that macrophages can secrete factors promoting angiogenesis and tumor growth (9). However, as illustrated by our in vitro Boyden chamber coculture experiments, protumorigenic properties could be unmasked when macrophage-to-tumor cell contact was denied. In these experiments, tumor cell migration, accompanied by nuclear translocation of ß-catenin, was instead stimulated by the presence of macrophages. It has previously been shown that murine macrophages can kill glioma cells transfected with the membrane but not the secreted, isoform of macrophage colony-stimulating factor in a phagocytosis-dependent process (32). Thus, the degree to which the antitumorigenic abilities manifest may partly depend on the abilities of macrophages to come in direct contact with tumor cells, as well as to achieve a high macrophage to cancer cell ratio.

Although colon cancer cells are not immune to protumorigenic properties of macrophages in vitro, antitumorigenic properties seem to dominate in the more complex situation in vivo, altogether resulting in a favorable prognosis in our clinical material. These results are consistent with some previous studies that have linked decreasing amounts of macrophages with more advanced-stage tumors in colorectal cancer patients (27, 28, 33). In an attempt to link macrophage infiltration to prognosis using material from 131 patients, CD68 infiltration alone did not correlate with prognosis (25). Somewhat surprisingly, however, the presence of both CD68 and vascular endothelial growth factor in tumor-infiltrating macrophages/stroma significantly improved prognosis (25). Our study established an association of CD68⁺ macrophages with improved prognosis. However, the question remains whether this reflects a predominant cytotoxic action of macrophages directly causing tumor regression, or whether it is more likely to reflect a gradual breakdown of immune responses eventually leading to tumor progression following a reduction in macrophage numbers, as proposed by Hakansson et al. (27). To that end, we have shown that under certain circumstances, PMA-activated U937 cells can inhibit the growth of some colon cancer cell lines. More significantly, it was recently reported that macrophage depletion in rats bearing colon cancer xenografts promoted enhanced cancer cell growth and impaired survival (34). Taken together, these results point to an important and direct role for macrophages in antitumor defense in colon cancer. Based on data presented herein, survival benefits require a high macrophage to cancer cell ratio, either along the whole tumor front or locally in "hotspots."

Along the tumor front, well-bordered, noninvasive tumor areas alternate with sites showing a more infiltrative growth pattern. Macrophage hotspots were often found at sites of infiltrative growth. Hauptmann et al. (35) have previously reported that 27E10-positive inflammatory macrophages were dominating at invasive areas and hypothesized that these macrophages could favor invasion. However, Hauptmann et al. did not assess prognosis in a clinical material. Given our results, an alternative possibility could be that a vigorous macrophage response (hotspot) at sites of ongoing invasion is an important feature of the protective action of macrophages that may delay or, in some cases, even prevent further tumor spread. It should also be noted that it was particularly at invasive sites, around small tumor nests, that a high macrophage to tumor cell ratio with efficient cell-to-cell contact was observed. The positive effect of massive infiltration was also reflected by the in vitro experiments allowing cell-to-cell contact, where colon cancer cell survival was reduced with increasing macrophage to cancer cell ratios. The highest cell ratios used in the coculture experiments corresponded roughly to those observed in hotspot grade 4. Thus, high macrophage infiltration is associated with improved prognosis in our clinical material, as well as in an animal model (34), and a high macrophage to tumor cell ratio inhibits cancer cell growth in vitro. Similarly, massive macrophage/monocyte infiltration was previously reported to be associated with tumor destruction, whereas a moderate infiltration resulted in growth of melanoma tumor xenografts (36).

Considering that we obtained a very strong correlation between lymphocytic and macrophage infiltration, lymphocytes seem to play an important role in the macrophage-promoted antitumorigenic defense. When staining a submaterial of 22 colorectal tumors, we found that CD20⁺ cells (B cells), when present, mainly showed a follicular staining pattern distant from the tumor front, whereas CD8⁺ cells, like CD68⁺ cells, were localized along the tumor front.⁴ High infiltration of CD8⁺ T cells tended to follow high infiltration CD68.⁴ Interestingly, whereas lymphocyte infiltration, as determined by routine H&E staining, was a strong prognostic marker in multivariate analysis when CD68TF_Hotspot was not included as a parameter, its prognostic value was lost if CD68TF_Hotspot was included. Combined with the finding that immune cell infiltrate was essentially absent in macrophage depleted colon tumor bearing rats (34), these results indicate that macrophages are essential for efficient recruitment of lymphocytes and that the interplay between macrophages and lymphocytes is of profound importance for an effective antitumor defense in colorectal cancer. The presence of high infiltration of both CD8⁺ and CD68⁺ cells has also been reported to positively influence prognosis in colon cancer patients (26), as well as the overall inflammatory cell reaction at the tumor front as evaluated in H&E-stained sections (29).

In conclusion, it is clear that monocytes/macrophages have protumorigenic as well as antitumorigenic properties in colon cancer. Assistance from T cells and direct macrophage-to-tumor cell contact may be required to manifest the antitumorigenic, or, alternatively, to counterbalance, the protumorigenic properties of macrophages in this cancer type. In addition, a high macrophage to cancer cell ratio increases the likelihood that the balance is shifted toward predominantly antitumorigenic properties, resulting in improved prognosis for colon cancer patients.

	Footnotes

 
Grant support: Swedish Cancer Society grant 2520-B05-19XBB; Lion's Cancer Research Foundation, Umeå University; and Vasterbotten County Council.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

⁴ Our unpublished observations.

Received 8/18/06; revised 11/ 6/06; accepted 11/20/06.

	References

Top Abstract Materials and Methods Results Discussion References

 

1. Coussens LM, Werb Z. Inflammation and cancer. Nature 2002;420:860–7.[CrossRef][Medline]
2. Pollard JW. Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer 2004;4:71–8.[CrossRef][Medline]
3. Urban JL, Shepard HM, Rothstein JL, Sugarman BJ, Schreiber H. Tumor necrosis factor: a potent effector molecule for tumor cell killing by activated macrophages. Proc Natl Acad Sci U S A 1986;83:5233–7.[Abstract/Free Full Text]
4. Cui S, Reichner JS, Mateo RB, Albina JE. Activated murine macrophages induce apoptosis in tumor cells through nitric oxide-dependent or -independent mechanisms. Cancer Res 1994;54:2462–7.[Abstract/Free Full Text]
5. Martin JH, Edwards SW. Changes in mechanisms of monocyte/macrophage-mediated cytotoxicity during culture. Reactive oxygen intermediates are involved in monocyte-mediated cytotoxicity, whereas reactive nitrogen intermediates are employed by macrophages in tumor cell killing. J Immunol 1993;150:3478–86.[Abstract]
6. Hansson M, Asea A, Ersson U, Hermodsson S, Hellstrand K. Induction of apoptosis in NK cells by monocyte-derived reactive oxygen metabolites. J Immunol 1996;156:42–7.[Abstract]
7. Onozaki K, Matsushima K, Kleinerman ES, Saito T, Oppenheim JJ. Role of interleukin 1 in promoting human monocyte-mediated tumor cytotoxicity. J Immunol 1985;135:314–20.[Abstract]
8. Fratelli M, Gagliardini V, Galli G, Gnocchi P, Ghiara P, Ghezzi P. Autocrine interleukin-1ß regulates both proliferation and apoptosis in EL4-6.1 thymoma cells. Blood 1995;85:3532–7.[Abstract/Free Full Text]
9. Leek RD, Harris AL. Tumor-associated macrophages in breast cancer. J Mammary Gland Biol Neoplasia 2002;7:177–89.[CrossRef][Medline]
10. Lewis JS, Landers RJ, Underwood JC, Harris AL, Lewis CE. Expression of vascular endothelial growth factor by macrophages is up-regulated in poorly vascularized areas of breast carcinomas. J Pathol 2000;192:150–8.[CrossRef][Medline]
11. Leek RD, Hunt NC, Landers RJ, Lewis CE, Royds JA, Harris AL. Macrophage infiltration is associated with VEGF and EGFR expression in breast cancer. J Pathol 2000;190:430–6.[CrossRef][Medline]
12. Arnott CH, Scott KA, Moore RJ, et al. Tumour necrosis factor- mediates tumour promotion via a PKC- and AP-1-dependent pathway. Oncogene 2002;21:4728–38.[CrossRef][Medline]
13. Lin EY, Gouon-Evans V, Nguyen AV, Pollard JW. The macrophage growth factor CSF-1 in mammary gland development and tumor progression. J Mammary Gland Biol Neoplasia 2002;7:147–62.[CrossRef][Medline]
14. Leek RD, Lewis CE, Whitehouse R, Greenall M, Clarke J, Harris AL. Association of macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma. Cancer Res 1996;56:4625–9.[Abstract/Free Full Text]
15. Lee AH, Happerfield LC, Bobrow LG, Millis relative risk. Angiogenesis and inflammation in invasive carcinoma of the breast. J Clin Pathol 1997;50:669–73.[Abstract/Free Full Text]
16. Lissbrant IF, Stattin P, Wikstrom P, Damber JE, Egevad L, Bergh A. Tumor associated macrophages in human prostate cancer: relation to clinicopathological variables and survival. Int J Oncol 2000;17:445–51.[Medline]
17. Hanada T, Nakagawa M, Emoto A, Nomura T, Nasu N, Nomura Y. Prognostic value of tumor-associated macrophage count in human bladder cancer. Int J Urol 2000;7:263–9.[CrossRef][Medline]
18. Nishie A, Ono M, Shono T, et al. Macrophage infiltration and heme oxygenase-1 expression correlate with angiogenesis in human gliomas. Clin Cancer Res 1999;5:1107–13.[Abstract/Free Full Text]
19. Salvesen HB, Akslen LA. Significance of tumour-associated macrophages, vascular endothelial growth factor and thrombospondin-1 expression for tumour angiogenesis and prognosis in endometrial carcinomas. Int J Cancer 1999;84:538–43.[CrossRef][Medline]
20. Fujimoto J, Sakaguchi H, Aoki I, Tamaya T. Clinical implications of expression of interleukin 8 related to angiogenesis in uterine cervical cancers. Cancer Res 2000;60:2632–5.[Abstract/Free Full Text]
21. Shimura S, Yang G, Ebara S, Wheeler TM, Frolov A, Thompson TC. Reduced infiltration of tumor-associated macrophages in human prostate cancer: association with cancer progression. Cancer Res 2000;60:5857–61.[Abstract/Free Full Text]
22. Koukourakis MI, Giatromanolaki A, Kakolyris S, et al. Different patterns of stromal and cancer cell thymidine phosphorylase reactivity in non-small-cell lung cancer: impact on tumour neoangiogenesis and survival. Br J Cancer 1998;77:1696–703.[Medline]
23. Kerr KM, Johnson SK, King G, Kennedy MM, Weir J, Jeffrey R. Partial regression in primary carcinoma of the lung: does it occur? Histopathology 1998;33:55–63.[Medline]
24. Migita T, Sato E, Saito K, et al. Differing expression of MMPs-1 and -9 and urokinase receptor between diffuse- and intestinal-type gastric carcinoma. Int J Cancer 1999;84:74–9.[CrossRef][Medline]
25. Khorana AA, Ryan CK, Cox C, Eberly S, Sahasrabudhe DM. Vascular endothelial growth factor, CD68, and epidermal growth factor receptor expression and survival in patients with stage II and stage III colon carcinoma: a role for the host response in prognosis. Cancer 2003;97:960–8.[CrossRef][Medline]
26. Funada Y, Noguchi T, Kikuchi R, Takeno S, Uchida Y, Gabbert HE. Prognostic significance of CD8+ T cell and macrophage peritumoral infiltration in colorectal cancer. Oncol Rep 2003;10:309–13.[Medline]
27. Hakansson L, Adell G, Boeryd B, Sjogren F, Sjodahl R. Infiltration of mononuclear inflammatory cells into primary colorectal carcinomas: an immunohistological analysis. Br J Cancer 1997;75:374–80.[Medline]
28. Nakayama Y, Nagashima N, Minagawa N, et al. Relationships between tumor-associated macrophages and clinicopathological factors in patients with colorectal cancer. Anticancer Res 2002;22:4291–6.[Medline]
29. Klintrup K, Makinen JM, Kauppila S, et al. Inflammation and prognosis in colorectal cancer. Eur J Cancer 2005;41:2645–54.[CrossRef][Medline]
30. Harris P, Ralph P. Human leukemic models of myelomonocytic development: a review of the HL-60 and U937 cell lines. J Leukoc Biol 1985;37:407–22.[Abstract]
31. Kitamura H, Nakagawa T, Takayama M, Kimura Y, Hijikata A, Ohara O. Post-transcriptional effects of phorbol 12-myristate 13-acetate on transcriptome of U937 cells. FEBS Lett 2004;578:180–4.[CrossRef][Medline]
32. Jadus MR, Williams CC, Avina MD, et al. Macrophages kill T9 glioma tumor cells bearing the membrane isoform of macrophage colony stimulating factor through a phagocytosis-dependent pathway. J Immunol 1998;160:361–8.[Abstract/Free Full Text]
33. Sickert D, Aust DE, Langer S, Haupt I, Baretton GB, Dieter P. Characterization of macrophage subpopulations in colon cancer using tissue microarrays. Histopathology 2005;46:515–21.[CrossRef][Medline]
34. Oosterling SJ, van der Bij GJ, Meijer GA, et al. Macrophages direct tumour histology and clinical outcome in a colon cancer model. J Pathol 2005;207:147–55.[CrossRef][Medline]
35. Hauptmann S, Zwadlo-Klarwasser G, Hartung P, Klosterhalfen B, Kirkpatrick CJ, Mittermayer C. Association of different macrophage phenotypes with infiltrating and non-infiltrating areas of tumor-host interface in colorectal carcinoma. Pathol Res Pract 1994;190:159–67.[Medline]
36. Nesbit M, Schaider H, Miller TH, Herlyn M. Low-level monocyte chemoattractant protein-1 stimulation of monocytes leads to tumor formation in nontumorigenic melanoma cells. J Immunol 2001;166:6483–90.[Abstract/Free Full Text]

This article has been cited by other articles:

				I. Espinosa, A. H. Beck, C.-H. Lee, S. Zhu, K. D. Montgomery, R. J. Marinelli, K. N. Ganjoo, T. O. Nielsen, C. B. Gilks, R. B. West, et al. Coordinate Expression of Colony-Stimulating Factor-1 and Colony-Stimulating Factor-1-Related Proteins Is Associated with Poor Prognosis in Gynecological and Nongynecological Leiomyosarcoma Am. J. Pathol., June 1, 2009; 174(6): 2347 - 2356. [Abstract] [Full Text] [PDF]

				M. Mehibel, S. Singh, E. C. Chinje, R. L. Cowen, and I. J. Stratford Effects of cytokine-induced macrophages on the response of tumor cells to banoxantrone (AQ4N) Mol. Cancer Ther., May 1, 2009; 8(5): 1261 - 1269. [Abstract] [Full Text] [PDF]

				B. Fingleton Tissue-Specific Regulation of Cancer Development by Immune Cells and Their Mediators Am. Assoc. Cancer Res. Educ. Book, April 18, 2009; 2009(1): 149 - 152. [Full Text] [PDF]

				S. Ogino, G. J. Kirkner, K. Nosho, N. Irahara, S. Kure, K. Shima, A. Hazra, A. T. Chan, R. Dehari, E. L. Giovannucci, et al. Cyclooxygenase-2 Expression Is an Independent Predictor of Poor Prognosis in Colon Cancer Clin. Cancer Res., December 15, 2008; 14(24): 8221 - 8227. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Forssell, J. Articles by Palmqvist, R. Search for Related Content PubMed PubMed Citation Articles by Forssell, J. Articles by Palmqvist, R.