Proc. Natl. Acad. Sci. USAVol. 86, pp. 5064-5068, July 1989Immunology

Regulation of tumor necrosis factor gene expression in colorectaladenocarcinoma: In vivo analysis by in situ hybridization

(immunohistochemistry)

STEFAN BEISSERT*, MICHAEL BERGHOLZt, INGE WAASE*, GERD LEPSIENt, ALFRED SCHAUERt,KLAUS PFIZENMAIER*, AND MARTIN KR6NKE*§*Kfinische Arbeitsgruppe, Max-Planck-Gesellschaft, Gosslerstrasse 10 d, 3400 Gdttingen, Federal Republic of Germany; and tZentrum Pathologie andtZentrum Chirurgie, Universitit G6ttingen, Robert-Koch-Strasse 40, 3400 Gottingen, Federal Republic of Germany

Communicated by Lloyd J. Old, March 9, 1989 (received for review December 12, 1988)

ABSTRACT Tumor necrosis factor (TNF) produced bymacrophages is thought to contribute to the host defenseagainst development of cancer. However, since tumor cellsthemselves are able to produce TNF, it is conceivable that TNFmay also play an adverse pathological role in carcinogenesis.To better understand the functional ignificance of TNF inneoplastic disease, we have determined the cellular source ofTNF activity produced in 10 patients with colorectal cancer.Northern blot analysis of RNAs extracted from fresh biopsyspecimens revealed detectable TNF mRNA levels in all in-stances. By using in situ hybridization of frozen sections,scattered cells expressing TNF mRNA could be discerned.Based on morphological criteria, these TNF-positive cells mostlikely belong to the macrophage lineage. Macrophages innormal tissue surrounding the tumor did not express TNFmRNA, suggesting that macrophage activation occurs locally atthe site of neoplastic transformatn. Immunohistochemistryusing anti-TNF monoclonal antibodies revealed that less than1% of tumor-infiltrating macrophages synthesize TNF protein.Thus we present evidence that in colorectal cancer only a smallproportion of tumor-infiltrating macrophages produces TNF,indicating that the microenvronment of the tumor providesadequate, yet suboptimal, conditions for macrophage activa-tion.

Tumor necrosis factor (TNF) has been originally described asa serum activity in lipopolysaccharide-treated mice that iscapable of causing hemorrhagic necrosis of animal tumors(1). However, since the availability of highly purified recom-binant TNF, evidence has accumulated indicating that TNFhas a wide range of biological activities, affecting the growth,differentiation, or function of virtually every cell type inves-tigated (2-5). Although the complexity of TNF activitiesmakes it at present difficult to assign a specific role to TNFin vivo, its direct tumoricidal activity and its role in inflam-mation are thought to contribute to the host's defense againsttumors. This proposal has been challenged by findings indi-cating that many cell types, including tumor cells themselves,are able to produce either TNF or lymphotoxin (also calledTNF-,B) (6-10). Lymphotoxin is a TNF-related cytokine thatbinds to the same receptor (11) and exerts similar biologicalactivities (12). TNF or lymphotoxin production by tumorcells suggests that these cytokines have a possible pathogenicrole in tumorigenesis. Indeed, the secretion of lymphotoxinby human myeloma cells has been linked to osteoclastic bonedestruction and hypercalcemia in patients with myeloma (8).In addition, because of its potent angiogenic activity (13, 14),TNF may stimulate the growth of blood vessels, therebypromoting tumor development. Furthermore, the ability of

TNF to induce collagenase synthesis (15) may lead to tissuedestruction facilitating the invasive growth of tumors. Thus,demonstration of TNF production in cancer may reflecteither protective or pathogenic processes. Clearly, a first stepto understanding the pathophysiological role of TNF incancer would be the identification of the TNF-producing celltype at the actual site of disease. Thus we analyzed samplesfrom 10 patients with colorectal carcinoma. We show thattumor-infiltrating macrophages and not tumor cells present incolorectal adenocarcinoma express TNF mRNA and pro-duce TNF protein.

MATERIALS AND METHODSRNA Blotting Analysis. Total cellular RNA was extracted

from fresh biopsies by the method of Chirgwin et al. (16), 30gg of RNA was electrophoresed on formaldehyde gels,transferred to nitrocellulose filters, and hybridized to aTNF-specific cDNA probe (an 800-base-pair EcoRI fragmentof A42-4) (17) or to an HLA-B7-specific cDNA (18) labeledwith [32P]dCTP by random priming. After hybridization,filters were washed and exposed to Kodak XAR films byusing an intensifying screen.In Situ Hybridization. In situ hybridization was performed

essentially as described by Hogan et al. (19). Freshly isolatedbiopsies were frozen immediately without any fixation. Spec-imen blocks were cut at 8 glm in a cryostat at -200C. Serialfrozen sections were collected on "subbed" slides (20), driedfor 10 min at 550C, and fixed in 4% (wt/vol) paraformalde-hyde. To remove proteins possibly associated with mRNA,slides were incubated in 2x SSC (lx SSC = 0.15 M NaCl/0.015 M sodium citrate, pH 7.0) for 30 min at 700C, rinsed inphosphate-buffered saline (PBS, 20 mM sodium phosphate/0.7% NaCl, pH 7.4), and digested with Pronase (0.2 mg/ml)for 10 min at room temperature. Slides were then refixed,acetylated, dehydrated, and finally air-dried.RNA probes were labeled by using SP6 or T7 RNA

polymerases and 35S-labeled UTP as described (21). Tem-plate TNF- and HLA-B7-specific DNAs were linearized bydigestion with enough restriction enzyme to generate 35S_labeled run-on transcripts 380-1100 bases long. Hybridiza-tion was performed at 500C in a humid chamber. Slides werewashed in 50o (vol/vol) formamide/2x SSC followed bydigestion with RNase A (20 gg/ml) and three further washesin 50o formamide/2x SSC. For autoradiography, air-driedslides were coated with NTB2 emulsion (Eastman Kodak)and exposed at 40C for 10 days. Slides were developed andcounter-stained with hematoxylin/eosin.

Immunohistochemistry. For immunostaining frozen sec-tions were incubated with monoclonal antibodies as indi-cated. Monoclonal anti-humanTNF antibody 154/6 was a gift

Abbreviation: TNF, tumor necrosis factor.§To whom reprint requests should be addressed.

5064

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Proc. Natl. Acad. Sci. USA 86 (1989) 5065

of G. Trinchieri (Philadelphia). Monoclonal antibody 63D3,recognizing a monocyte-specific antigen (22), was purchasedfrom the American Type Culture Collection. Alkaline phos-phatase-anti-alkaline phosphatase complexes were preparedas described by Cordell et al. (23). Sections were counter-stained with hematoxylin/eosin.

RESULTSTNFmRNA Expression in Colorectal Cancer. Tumor biopsy

specimens were obtained from 10 patients with colorectalcancer during abdominal surgery. Total RNA was extractedand analyzed for TNF mRNA levels. As shown in Fig. 1, theabundance of TNF mRNA varied depending on the tumorspecimen investigated. Longer exposure of the RNA blotrevealed that, indeed, TNF mRNA could be detected in allinstances (data not shown). The tumor ofpatient 7 apparentlyexpressed the highest level ofTNF mRNA. Control hybrid-ization to a HLA-A,B,C-specific cDNA probe confirmed theintegrity of the RNA samples loaded. The different HLA-A,B,C mRNA levels observed most likely reflect variableHLA-A,B,C gene expression by individual tumor tissuessince control ethidium bromide staining indicated equivalentquantities of RNA transferred to nitrocellulose filters.In Vivo TNF mRNA Expression by Tumor-Infiltrating Mac-

rophages. To determine the cellular source of TNF mRNA,in situ hybridization of frozen tumor sections was performedusing a 35S-labeled antisense TNF RNA probe. As exempli-fied by patient 7, the majority of cells have 5-10 grains percell (Fig. 2a) corresponding to the background staining re-vealed by hybridization to a 35S-labeled sense HLA-B7 RNAprobe (Fig. 2c). Only a few scattered cells had grain countsthat were well above background (40-200 grains), which wasconsidered specific hybridization to TNF mRNA. By histo-morphological criteria (azurophilic cytoplasm, large lobularnucleus, and dense chromatin), these cells were consideredto be of the macrophage/monocyte lineage.To demonstrate the sensitivity of the in situ hybridization,

we used an 35S-labeled antisense HLA-B7 RNA probe. Asshown in Fig. 2b, greater than 80%o of cells were positive,indicating that most if not all of the cells contained intactmRNA that was accessible for effective hybridization. Similarresults were obtained when tumor specimens from nine otherpatients were analyzed by in situ hybridization (Table 1).

Tumor-Inflltrating Macrophages Produce TNF Protein.Since TNF gene expression is largely regulated at a post-transcriptional level-that is, translation ofTNF mRNA andsecretion of TNF require additional stimuli (10, 24, 25)-itwas important to determine whether TNF mRNA-positivemacrophages actually produce TNF protein. As shown inFig. 3, immunohistochemistry using an anti-TNF monoclonal

1 2 3 4 5 6 7 8 9 10cDNA probe

40 pTNF

o* it Sp pHLA-B7

FIG. 1. TNF mRNA in tumor biopsy specimens of 10 patientswith colorectal carcinoma. Total RNA (30 tg per lane) was size-separated in 1% agarose/formaldehyde gels, transferred to nitrocel-lulose filters, and hybridized to 32P-labeled TNF and HLA-B7-specific cDNA probes.

FIG. 2. In vivo TNF mRNA expression in the adenocarcinomafrom patient 7. Frozen sections (8 ,gm thick) were hybridized to35S-labeled antisense TNF (a) or HLA-B7 (b) complementary RNAprobes. Control hybridization was performed with a sense HLA-B7RNA probe (c). After a 6-day exposure, slides were developed andcounterstained with hematoxylin/eosin. (For a, x400; for b and c,x250.)

antibody strongly stained cells with macrophage-like mor-phology, indicating that tumor-infiltrating macrophages doproduce TNF protein in vivo.Lack of TNF-Producing Macrophages in Normal Tissue

Surrounding the Tumor. Normal colorectal tissue of patient7 adjacent to the site of tumor had strong mononuclearinfiltration (Fig. 4a) comparable to that observed in theadenocarcinoma (Fig. 5a). However, neither TNF mRNAnor TNF protein could be detected by in situ hybridization(Fig. 4b) or by immunostaining (data not shown), respec-tively. This finding indicates that it is the microenvironmentof colorectal cancer that provides the stimuli for the regionalactivation of macrophages.

Activation Status of Tumor-Infiltrating Macrophages. Todetermine the level of macrophage activation in colorectalcancer in further detail, we compared the extent of tumor

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Proc. Nati. Acad. Sci. USA 86 (1989)

Table 1. In situ analysis of TNF production in colorectal cancerSite of Frequency of TNF mRNA- TNF-adeno- infiltrating positive producing

Patient carcinoma macrophages,* % cells,t % cells,*%1 Sigmoid colon 15-25 2 0.22 Sigmoid colon 18-35 3 0.33 Colon 20-30 1.5 0.24 Colon 15-25 2 0.35 Sigmoid colon 30-40 1.5 0.16 Sigmoid colon 25-35 1.5 0.17 Rectum 25-35 6 0.38 Rectum 25-35 2 0.19 Colon 20-30 1 0.110 Rectum 30-40 <1 0

*Cryostat sections of tumor biopsy specimens were immunostainedwith the monocytic-specific antibody 63D3. Frequencies of infil-trating macrophages were revealed by microscopic evaluation ofseveral visual fields corresponding to more than 1000 nucleatedcells. Data are the ranges ofmonocyte infiltration that varied withina given tumor specimen depending on the particular section inves-tigated.tTNF mRNA-positive cells were visualized by in situ hybridizationwith an antisense TNF complementary RNA probe. Frequencies ofTNF mRNA-positive cells were estimated by evaluating >2000cells.tTNF protein-producing cells were detected by immunostaining withanti-TNF antibody. Frequencies of positive cells were estimated byevaluating >2000 cells.

infiltration by macrophages with the frequency of macro-phages expressing TNF mRNA or producing TNF protein.As shown in Fig. Sa, approximately 30%o of the cells in therectal adenocarcinoma of patient 7 had macrophage-likemorphology and were stained with 63D3, a monocyte-specific monoclonal antibody (22). Bright- and dark-fieldmicroscopic evaluation of the in situ hybridization using35S-labeled antisense TNF RNA probe showed that =6% ofthe cells were TNF mRNA-positive (Fig. 5 c and d). Whenimmunostained by the anti-TNF monoclonal antibody, only0.3% of the mononuclear cells were positive (Fig. 5b andTable 1). Thus the data indicate that approximately everyfifth tumor-infiltrating macrophage only expresses TNFmRNA and even fewer synthesize detectable levels ofTNFprotein. Similar results were obtained from correspondinganalyses of nine other patients with colorectal adenocarci-noma (Table 1).

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FIG. 3. In vivo TNF production in rectal adenocarcinoma.Cryostat sections from the adenocarcinoma of patient 7 were immnu-nostained with monoclonal anti-TNF-antibody 154/6 using the alka-line phosphatase-anti-alkaline phosphatase method. Parallel sectionsanalyzed with isotype-matched control immunoglobulins were notstained. (x600.)

FIG. 4. Lack of TNF mRNA-positive cells in normal tissue.Cryostat sections of normal rectal tissue from patient 7 in theimmediate vicinity ofthe tumor were immunostained with monocyte-specific monoclonal antibody 63D3 (a) or hybridized to a 35S-labeledanti-sense TNF RNA probe (b). Arrows indicate macrophagesidentified by morphological criteria. (x250.)

DISCUSSIONTNF mRNA has been detected (26) in RNA samples ex-tracted from either peripheral blood lymphocytes or solidtumor tissue from cancer patients. Determination of eitherTNF activity in the serum or TNF mRNA levels in the RNAextracted from heterogenous tumor tissue, however, doesnot answer the crucial questions as to the cellular source andfunctional significance of TNF production in neoplastic dis-ease. In the present study, we have attempted to characterizethe conditions of TNF production in colorectal cancer. Weshow by in situ hybridization and immunohistochemistry thatin all cases of colorectal carcinoma investigated, TNF wasproduced by infiltrating macrophages. The observation thatthe mononuclear infiltrate expresses TNF mRNA in 10 out of10 colorectal carcinoma patients suggests that in this tumorTNF production may be a defense reaction executed byactivated macrophages.One of the principal findings of this report is that only a

fraction of tumor-infiltrating macrophages expresses TNFmRNA, and even fewer are able to produce TNF protein.Although it appears to be difficult to precisely judge thesensitivity of the in situ hybridization and immunostainingprocedures, analogous analyses of renal cell carcinoma spec-imens have shown that it is possible to demonstrate TNFmRNA as well as TNF protein in greater than 50%o of thetumor-infiltrating macrophages (I.W. and M.K., unpublisheddata). Thus, the frequencies of TNF-positive macrophagesobserved might be only slightly underestimated. The quan-titative representation of tumor-infiltrating macrophages ex-

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Proc. Natl. Acad. Sci. USA 86 (1989) 5067

FIG. 5. Activation status of tumor-infiltrating macrophages. Frozen sections from patient 7 were immunostained with either monocyte-specific monoclonal antibody 63D3 (a) or monoclonal anti-TNF antibody 154/6 (b). In situ hybridization was performed using antisense TNF(c) and HLA-B7 (e) RNA probes. (d) Dark-field image of c. (f) Dark-field image of e. Arrows indicate TNF protein- (b) or TNF mRNA-positive(c) cells. (x1OO.)

pressing TNF mRNA or TNF protein detected in this studysuggests that the control mechanisms of TNF gene expres-sion defined in vitro are recapitulated in vivo. It has beenshown that murine macrophages require a sequence of twodistinct signals to become activated for cellular toxicity (27,28), which may be TNF-mediated (29, 30). The first signalserves as a "priming" factor and the second signal triggersthe final activation. The priming signal may mediate tran-scriptional activation of the TNF gene, and the second signalmay be required to allow translation of the protein and TNFsecretion by macrophages (24, 25, 31, 32). As illustrated inthis report, the number of detectable TNF-producing mac-rophages is quite small, even though the tumor is heavilyinfiltrated. This suggests that the population of tumor-infiltrating macrophages is only suboptimally activated. Ac-tivation of macrophages apparently occurs at the site of

neoplastic transformation, since macrophages present in thenormal tissue in the immediate vicinity of the tumor neitherexpress TNF mRNA nor secrete TNF protein. It is thereforeconcluded that the colorectal tumor itself provides the mi-croenvironment for macrophage activation.Our findings have implications for entering patients with

colorectal cancer into clinical trials with biological responsemodifiers. Since, in this tumor, the majority of infiltratingmacrophages seems to be suboptimally activated, treatmentwith recombinant TNF may supplement the host's defenseagainst tumor progression and eventually benefit the patient.Furthermore, treatment with macrophage-activating agents,such as the interferons, might enhance the endogenousantitumoral activities of the mononuclear infiltrate.

We thank G. Trinchieri (Wistar Institute, Philadelphia) for pro-

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Proc. Natl. Acad. Sci. USA 86 (1989)

viding anti-TNF monoclonal antibody 154/6. We thank A. Hofsom-mer, B. Junemann, and M. Kollhof for technical assistance and G.Schmidt for preparing the manuscript. We also thank Drs. P. Grussand G. Dresslerfor helpful discussions. This work was done in partialfulfillment of the M.D. thesis for S.B. and I.W.

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