Intratumoral Heterogeneity for Expression of Tyrosine Kinase Growth Factor Receptors in Human Colon Cancer Surgical Specimens and Orthotopic Tumors
2008; Elsevier BV; Volume: 172; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2008.070625
ISSN1525-2191
AutoresToshio Kuwai, Tõru Nakamura, Sun-Jin Kim, Takamitsu Sasaki, Yasuhiko Kitadai, Robert R. Langley, Dominic Fan, Stanley R. Hamilton, Isaiah J. Fidler,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoThe design of targeted therapy, particularly patient-specific targeted therapy, requires knowledge of the presence and intratumoral distribution of tyrosine kinase receptors. To determine whether the expression of such receptors is constant or varies between and within individual colon cancer neoplasms, we examined the pattern of expression of the ligands, epidermal growth factor, vascular endothelial growth factor, and platelet-derived growth factor-B as well as their respective receptors in human colon cancer surgical specimens and orthotopic human colon cancers growing in the cecal wall of nude mice. The expression of the epidermal growth factor receptor and the vascular endothelial growth factor receptor on tumor cells and stromal cells, including tumor-associated endothelial cells, was heterogeneous in surgical specimens and orthotopic tumors. In some tumors, the receptor was expressed on both tumor cells and stromal cells, and in other tumors the receptor was expressed only on tumor cells or only on stromal cells. In contrast, the platelet-derived growth factor receptor was expressed only on stromal cells in both surgical specimens and orthotopic tumors. Examination of receptor expression in both individual surgical specimens and orthotopic tumors revealed that the platelet-derived growth factor receptor was expressed only on stromal cells and that the patterns of epidermal growth factor receptor and vascular endothelial growth factor receptor 2 expression differed between tumor cells. This heterogeneity in receptor expression among different tumor cells suggests that targeting a single tyrosine kinase may not yield eradication of the disease. The design of targeted therapy, particularly patient-specific targeted therapy, requires knowledge of the presence and intratumoral distribution of tyrosine kinase receptors. To determine whether the expression of such receptors is constant or varies between and within individual colon cancer neoplasms, we examined the pattern of expression of the ligands, epidermal growth factor, vascular endothelial growth factor, and platelet-derived growth factor-B as well as their respective receptors in human colon cancer surgical specimens and orthotopic human colon cancers growing in the cecal wall of nude mice. The expression of the epidermal growth factor receptor and the vascular endothelial growth factor receptor on tumor cells and stromal cells, including tumor-associated endothelial cells, was heterogeneous in surgical specimens and orthotopic tumors. In some tumors, the receptor was expressed on both tumor cells and stromal cells, and in other tumors the receptor was expressed only on tumor cells or only on stromal cells. In contrast, the platelet-derived growth factor receptor was expressed only on stromal cells in both surgical specimens and orthotopic tumors. Examination of receptor expression in both individual surgical specimens and orthotopic tumors revealed that the platelet-derived growth factor receptor was expressed only on stromal cells and that the patterns of epidermal growth factor receptor and vascular endothelial growth factor receptor 2 expression differed between tumor cells. This heterogeneity in receptor expression among different tumor cells suggests that targeting a single tyrosine kinase may not yield eradication of the disease. Growth factors and their receptors play a major role in the regulation of progressive cancer growth and metastasis.1Ullrich A Schlessinger J Signal transduction by receptors with tyrosine kinase activity.Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4569) Google Scholar, 2Yarden Y Ullrich A Growth factor receptor tyrosine kinases.Annu Rev Biochem. 1988; 57: 443-478Crossref PubMed Scopus (1380) Google Scholar For this reason, the expression of growth factors and their receptors by cancer cells and host stromal cells, including tumor-associated endothelial cells, has been an attractive target for therapy.3Kim SJ Uehara H Yazici S Langley RR He J Tsan R Fan D Killion JJ Fidler IJ Simultaneous blockade of platelet-derived growth factor-receptor and epidermal growth factor-receptor signaling and systemic administration of paclitaxel as therapy for human prostate cancer metastasis in bone of nude mice.Cancer Res. 2004; 64: 4201-4208Crossref PubMed Scopus (97) Google Scholar, 4Yokoi K Sasaki T Bucana CD Fan D Baker CH Kitadai Y Kuwai T Abbruzzese JL Fidler IJ Simultaneous inhibition of EGFR, VEGFR, and platelet-derived growth factor receptor signaling combined with gemcitabine produces therapy of human pancreatic carcinoma and prolongs survival in an orthotopic nude mouse model.Cancer Res. 2005; 65: 10371-10380Crossref PubMed Scopus (89) Google Scholar Recently, specific inhibitors of tyrosine kinase receptors have been targeted in patients with chemotherapy-refractory neoplasms, and this approach has led to prolongation of median survival in some patients.5Herbst RS Maddox AM Rothenberg ML Small EJ Rubin EH Baselga J Rojo F Hong WK Swaisland H Averbuch SD Ochs J LoRusso PM Selective oral epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 is generally well-tolerated and has activity in non-small-cell lung cancer and other solid tumors: results of a phase I trial.J Clin Oncol. 2002; 20: 3815-3825Crossref PubMed Scopus (548) Google Scholar, 6Mathew P Thall PF Jones D Perez C Bucana C Troncoso P Kim SJ Fidler IJ Logothetis C Platelet-derived growth factor receptor inhibitor imatinib mesylate and docetaxel: a modular phase I trial in androgen-independent prostate cancer.J Clin Oncol. 2004; 22: 3323-3329Crossref PubMed Scopus (111) Google Scholar, 7Strumberg D Richly H Hilger RA Schleucher N Korfee S Tewes M Faghih M Brendel E Voliotis D Haase CG Schwartz B Awada A Voigtmann R Scheulen ME Seeber S Phase I clinical and pharmacokinetic study of the novel Raf kinase and vascular endothelial growth factor receptor inhibitor BAY 43–9006 in patients with advanced refractory solid tumors.J Clin Oncol. 2005; 23: 965-972Crossref PubMed Scopus (787) Google Scholar The failure of this approach to produce improved clinical outcome in a larger proportion of the treated patients indicates the necessity of establishing better criteria for patient selection and evaluation of therapy. Tumor progression, angiogenesis, and metastasis are regulated by the interaction of tumor cells with the host organ microenvironment.8Fidler IJ Kim SJ Langley RR The role of the organ microenvironment in the biology and therapy of cancer metastasis.J Cell Biochem. 2007; 101: 927-936Crossref PubMed Scopus (113) Google Scholar The expression of growth factors and their receptors varies in different zones of any given organ and may differ among different cells of the same tumor.9Fidler IJ The pathogenesis of cancer metastasis: the “seed and soil” hypothesis revisited.Nat Rev Cancer. 2003; 3: 453-458Crossref PubMed Scopus (3438) Google Scholar, 10Fidler IJ The organ microenvironment and cancer metastasis.Differentiation. 2002; 70: 498-505Crossref PubMed Scopus (340) Google Scholar Overexpression of transforming growth factor-α (TGF-α), epidermal growth factor (EGF), and the EGF receptor (EGFR) has been reported to be associated with poor prognosis in multiple neoplasms.11Uhlman DL Nguyen P Manicel JC Zhang G Hagen K Fraley E Aeppli D Niehans GA Epidermal growth factor receptor and transforming growth factor α expression in papillary and non papillary renal cell carcinoma: correlation with metastatic behavior and prognosis.Clin Cancer Res. 1995; 1: 913-920PubMed Google Scholar, 12Atlas I Mendelsohn J Baselga J Fair WR Masui H Kumar R Growth regulation of human renal carcinoma cells: role of transforming growth factor α.Cancer Res. 1992; 52: 3335-3339PubMed Google Scholar, 13Moch H Sauter G Buchholz N Gasser TC Bubendorf L Waldman FM Mihatsch MJ Epidermal growth factor receptor expression is associated with rapid tumor cell proliferation and renal cell carcinoma.Hum Pathol. 1997; 11: 1255-1259Abstract Full Text PDF Scopus (90) Google Scholar, 14Hofmockel G Riess S Bassukas ID Dammrich J Epidermal growth factor family and renal cell carcinoma: expression and prognostic impact.Eur Urol. 1997; 4: 478-484Google Scholar, 15Yoshida K Tosaka A Takeuchi S Kobayashi N Epidermal growth factor receptor content in human renal cell carcinomas.Cancer. 1994; 73: 1913-1918Crossref PubMed Scopus (51) Google Scholar Similarly, expression of vascular endothelial growth factor (VEGF) is associated with increased vascular permeability, cell proliferation, and survival16Leung DW Cachianes G Kuang WJ Goeddel DV Ferrara N Vascular endothelial growth factor is a secreted angiogenic mitogen.Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4396) Google Scholar, 17Yuan F Chen Y Dellian M Safabakhsh N Ferrara N Jain RK Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody.Proc Natl Acad Sci USA. 1996; 93: 14765-14770Crossref PubMed Scopus (597) Google Scholar, 18Gerber HP McMurtrey A Kowalski J Yan M Keyt BA Dixit V Ferrara N Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3′-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation.J Biol Chem. 1998; 273: 30336-30343Crossref PubMed Scopus (1722) Google Scholar, 19Gerber HP Dixit V Ferrara N Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells.J Biol Chem. 1998; 273: 13313-13316Crossref PubMed Scopus (825) Google Scholar of endothelial cells. The expression of VEGF has been correlated with microvessel density of neoplasms,20Takahashi Y Kitadai Y Bucana CD Cleary KR Ellis LM Expression of vascular endothelial growth factor and its receptor. KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer.Cancer Res. 1995; 55: 3964-3968PubMed Google Scholar, 21Ferrara N Alitalo K Clinical applications of angiogenic growth factors and their inhibitors.Nat Med. 1999; 5: 1359-1364Crossref PubMed Scopus (906) Google Scholar metastasis, and, hence, poor prognosis.22Kuwai T Kitadai Y Tanaka S Onogawa S Matsutani N Kaio E Ito M Chayama K Expression of hypoxia-inducible factor-1alpha is associated with tumor vascularization in human colorectal carcinoma.Int J Cancer. 2003; 105: 176-181Crossref PubMed Scopus (154) Google Scholar, 23Ikeda N Adachi M Taki T Huang C Hashida H Takabayashi A Sho M Nakajima Y Kanehiro H Hisanaga M Nakano H Miyake M Prognostic significance of angiogenesis in human pancreatic cancer.Br J Cancer. 1999; 79: 1553-1563Crossref PubMed Scopus (289) Google Scholar, 24Ferrara N Davis-Smyth T The biology of vascular endothelial growth factor.Endocr Rev. 1997; 18: 4-25Crossref PubMed Scopus (3664) Google Scholar, 25Volm M Koomagi R Mattern J Prognostic value of vascular endothelial growth factor and its receptor Flt-1 in squamous cell lung cancer.Int J Cancer. 1997; 74: 64-68Crossref PubMed Scopus (236) Google Scholar, 26Strohmeyer D Rossing C Bauerfeind A Kaufmann O Schlechte H Bartsch G Loening S Vascular endothelial growth factor and its correlation with angiogenesis and p53 expression in prostate cancer.Prostate. 2000; 45: 216-224Crossref PubMed Scopus (99) Google Scholar The expression of platelet-derived growth factor (PDGF) and its receptor (PDGFR) by tumor cells, tumor-associated endothelial cells,3Kim SJ Uehara H Yazici S Langley RR He J Tsan R Fan D Killion JJ Fidler IJ Simultaneous blockade of platelet-derived growth factor-receptor and epidermal growth factor-receptor signaling and systemic administration of paclitaxel as therapy for human prostate cancer metastasis in bone of nude mice.Cancer Res. 2004; 64: 4201-4208Crossref PubMed Scopus (97) Google Scholar, 27Uehara H Kim SJ Karashima T Shepherd DL Fan D Tsan R Killion JJ Logothetis C Mathew P Fidler IJ Effects of blocking platelet-derived growth factor-receptor signaling in a mouse model of experimental prostate cancer bone metastases.J Natl Cancer Inst. 2003; 95: 458-470Crossref PubMed Scopus (252) Google Scholar, 28Kim SJ Uehara H Yazici S Busby JE Nakamura T He J Maya M Logothetis C Mathew P Wang X Do KA Fan D Fidler IJ Targeting platelet-derived growth factor receptor on endothelial cells of multidrug-resistant prostate cancer.J Natl Cancer Inst. 2006; 98: 783-793Crossref PubMed Scopus (89) Google Scholar and pericytes and myofibroblasts4Yokoi K Sasaki T Bucana CD Fan D Baker CH Kitadai Y Kuwai T Abbruzzese JL Fidler IJ Simultaneous inhibition of EGFR, VEGFR, and platelet-derived growth factor receptor signaling combined with gemcitabine produces therapy of human pancreatic carcinoma and prolongs survival in an orthotopic nude mouse model.Cancer Res. 2005; 65: 10371-10380Crossref PubMed Scopus (89) Google Scholar, 29Kim SJ Uehara H Yazici S He J Langley RR Mathew P Fan D Fidler IJ Modulation of bone microenvironment with zoledronate enhances the therapeutic effects of STI571 and paclitaxel against experimental bone metastasis of human prostate cancer.Cancer Res. 2005; 65: 3707-3715Crossref PubMed Scopus (56) Google Scholar, 30Kitadai Y Sasaki T Kuwai T Nakamura T Bucana CD Hamilton SR Fidler IJ Expression of activated platelet-derived growth factor receptor in stromal cells of human colon carcinomas is associated with metastatic potential.Int J Cancer. 2006; 119: 2567-2574Crossref PubMed Scopus (79) Google Scholar, 31Lindmark G Sundberg C Glimelius B Pahlman L Rubin K Gerdin B Stromal expression of platelet-derived growth factor beta-receptor and platelet-derived growth factor B-chain in colorectal cancer.Lab Invest. 1993; 69: 682-689PubMed Google Scholar, 32Sundberg C Ljungstrom M Lindmark G Gerdin B Rubin K Microvascular pericytes express platelet-derived growth factor-beta receptors in human healing wounds and colorectal adenocarcinoma.Am J Pathol. 1993; 143: 1377-1388PubMed Google Scholar is common to many neoplasms. Our recent data clearly established that in clinical specimens of human colon carcinomas, although PDGF is produced by tumor cells, the expression of PDGFR is restricted to stromal cells, including tumor-associated endothelial cells.30Kitadai Y Sasaki T Kuwai T Nakamura T Bucana CD Hamilton SR Fidler IJ Expression of activated platelet-derived growth factor receptor in stromal cells of human colon carcinomas is associated with metastatic potential.Int J Cancer. 2006; 119: 2567-2574Crossref PubMed Scopus (79) Google Scholar The significance of these findings is that tumor-associated stromal cells generate a microenvironment favorable to the survival and progressive growth of tumor cells.33Risau W Drexler H Mironov V Smits A Siegbahn A Funa K Heldin CH Platelet-derived growth factor is angiogenic in vivo.Growth Factors. 1992; 7: 261-266Crossref PubMed Scopus (230) Google Scholar For example, PDGFR signaling is related to recruitment of pericytes and control of interstitial fluid pressure,33Risau W Drexler H Mironov V Smits A Siegbahn A Funa K Heldin CH Platelet-derived growth factor is angiogenic in vivo.Growth Factors. 1992; 7: 261-266Crossref PubMed Scopus (230) Google Scholar, 34Ostman A PDGF receptors-mediators of autocrine tumor growth and regulators of tumor vasculature and stroma.Cytokine Growth Factor Rev. 2004; 15: 275-286Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 35Bergers G Song S Meyer-Morse N Bergsland E Hanahan D Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors.J Clin Invest. 2003; 111: 1287-1295Crossref PubMed Scopus (1218) Google Scholar, 36Pietras K Rubin K Sjoblom T Buchdunger E Sjoquist M Heldin CH Ostman A Inhibition of PDGF receptor signaling in tumor stroma enhances antitumor effect of chemotherapy.Cancer Res. 2002; 62: 5476-5484PubMed Google Scholar favorable to the survival and progressive growth of neoplasms.37Liotta LA Kohn EC The microenvironment of the tumour-host interface.Nature. 2001; 411: 375-379Crossref PubMed Scopus (2007) Google Scholar The design of targeted therapy directed against tyrosine kinase receptors, particularly the design of patient-specific therapy (“customization”), requires identification of the presence and intratumoral distribution of tyrosine kinase receptors. To determine whether the expression of these receptors is constant or varies between and within different colon neoplasms, we examined the expression of the ligands TGF-α, EGF, VEGF, and PDGF-B and their respective receptors in surgical specimens from several human colon cancers of different pathological stages and in orthotopic human colon cancers growing in nude mice. Our finding of intratumoral heterogeneity in receptor expression dictates the use of multiple tyrosine kinase inhibitors for effective therapy. Twelve surgical specimens of colon carcinoma (seven cases Duke's stage B; two cases Duke's stage C; and three cases Duke's stage D) were obtained from The University of Texas M. D. Anderson Cancer Center Tissue Bank. The specimens were frozen in liquid nitrogen or fixed in neutralized-buffered formalin within 30 minutes after surgical resection, making the analysis of phosphorylated receptors possible. The approval of the Institutional Review Board was obtained before we conducted the study. The highly metastatic human colon cancer cell lines KM12SM38Morikawa K Walker SM Jessup JM Fidler IJ In vivo selection of highly metastatic cells from surgical specimens of different primary human colon carcinoma implanted into nude mice.Cancer Res. 1988; 48: 1943-1948PubMed Google Scholar and HT294Yokoi K Sasaki T Bucana CD Fan D Baker CH Kitadai Y Kuwai T Abbruzzese JL Fidler IJ Simultaneous inhibition of EGFR, VEGFR, and platelet-derived growth factor receptor signaling combined with gemcitabine produces therapy of human pancreatic carcinoma and prolongs survival in an orthotopic nude mouse model.Cancer Res. 2005; 65: 10371-10380Crossref PubMed Scopus (89) Google Scholar, 30Kitadai Y Sasaki T Kuwai T Nakamura T Bucana CD Hamilton SR Fidler IJ Expression of activated platelet-derived growth factor receptor in stromal cells of human colon carcinomas is associated with metastatic potential.Int J Cancer. 2006; 119: 2567-2574Crossref PubMed Scopus (79) Google Scholar were maintained in minimal essential medium supplemented with 10% fetal bovine serum, sodium pyruvate, nonessential amino acids, l-glutamine, a twofold vitamin solution (Life Technologies, Grand Island, NY), and penicillin/streptomycin (Flow Laboratories, Rockville, MD). Adherent monolayer cultures were maintained at 37°C in a mixture of 5% CO2 and 95% air. The cultures were free of Mycoplasma species and pathogenic murine viruses (assayed by Science Applications International Co., Frederick, MD). The cultures were maintained for no longer than 12 weeks after recovery from frozen stock. The primary antibodies used were rabbit anti-phosphorylated VEGFR1 (Flk-1, Oncogene, Boston, MA), rabbit anti-VEGFR2 (C1158, Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-VEGFR2 (A3, Santa Cruz Biotechnology), rabbit anti-VEGF (A20, Santa Cruz Biotechnology), rabbit anti-phosphorylated EGFR (pEGFR, Tyr1173, Biosource, Camarillo, CA), mouse anti-EGFR (Zymed, San Francisco, CA), rabbit anti-EGFR (Santa Cruz Biotechnology), rabbit anti-TGF-α (Santa Cruz Biotechnology), rabbit anti-EGF (Santa Cruz Biotechnology), rabbit anti-PDGFR-β (Santa Cruz Biotechnology), rabbit anti-phosphorylated PDGFR-β (Santa Cruz Biotechnology), rabbit anti-PDGF-B (Santa Cruz Biotechnology), rat anti-mouse CD31 (BD PharMingen, San Diego, CA), and mouse anti-human CD31 (Dako A/S, Copenhagen, Denmark). Peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was a secondary antibody used for colorimetric immunohistochemical analysis. Fluorescent secondary antibodies were Cy3-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories), Cy5-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories), and Cy5-conjugated goat anti-rat IgG (Jackson ImmunoResearch Laboratories). Male athymic nude mice (NCI-nu) were purchased from the Animal Production Area of the National Cancer Institute Frederick Cancer Research and Development Center (Frederick, MD). The mice were housed and maintained under specific pathogen-free conditions in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the U.S. Department of Agriculture, U.S. Department of Health and Human Services, and the National Institutes of Health. The mice were used in accordance with institutional guidelines when they were 8 to 12 weeks old. To produce cecal tumors, KM12SM or HT29 cells were harvested from subconfluent cultures by a brief exposure to 0.25% trypsin and 0.02% EDTA. Trypsinization was stopped with medium containing 10% fetal bovine serum, and the cells were washed once in serum-free medium and resuspended in Ca2+/Mg2+-free Hanks' balanced salt solution. Only suspensions consisting of single cells with >90% viability were used. KM12SM cells (5 × 105) and HT29 cells (1 × 106) in 50 μl of Hanks' balanced salt solution were injected into the cecal wall of nude mice under a dissecting microscope as described previously.38Morikawa K Walker SM Jessup JM Fidler IJ In vivo selection of highly metastatic cells from surgical specimens of different primary human colon carcinoma implanted into nude mice.Cancer Res. 1988; 48: 1943-1948PubMed Google Scholar Six weeks after intracecal injection of tumor cells, mice were euthanized with methoxyflurane and necropsied. Body weight, tumor incidence, tumor weight, and incidence of lymph node metastasis were recorded. Tumor tissues were fixed in 10% neutralized buffered formalin to prepare paraffin sections or embedded in ornithine carbamyl transferase compound (Miles, Elkhart, IN) to prepare frozen sections for hematoxylin and eosin staining and immunohistochemical analysis. Paraffin-embedded tissues were used for immunohistochemical analyses of TGF-α, EGF, VEGF, and PDGF-B. The sections were deparaffinized in xylene and rehydrated with alcohol and phosphate-buffered saline (PBS). Endogenous peroxidase was blocked with 3% hydrogen peroxide in PBS. The slides were placed in a humidified chamber and incubated with protein blocking solution (5% normal horse serum and 1% normal goat serum in PBS) for 20 minutes at room temperature and incubated overnight at 4°C with primary antibody for TGF-α (1:00), EGF (1:00), VEGF (1:50), or PDGF-B (1:100). Slides were washed with PBS for 3 minutes three times and incubated for 1 hour with peroxidase-conjugated secondary antibody (1:500). Positive reaction was detected by exposure to stable 3,3′-diaminobenzidine (Phoenix Biotechnologies, Huntsville, AL). The slides were counterstained with Gill's no. 3 hematoxylin. Slides stained by diaminobenzidine or hematoxylin and eosin were examined in a Nikon Microphoto-FX microscope equipped with a three-chip charge-coupled device color video camera (model DXC990, Sony Corp., Tokyo, Japan). Digital images were captured using Optimas image analysis software (Media Cybernetics, Silver Spring, MD). Frozen sections of human colon cancers from the cecum of nude mice were assayed. Specimens were cut into 4-μm sections, mounted on positively charged slides, and stored at −80°C. Slides were fixed for 10 minutes in cold acetone, placed in a light-shielded humidified chamber, incubated for 20 minutes with protein blocking solution (5% normal horse serum and 1% normal goat serum in PBS) at room temperature, and incubated overnight at 4°C with primary antibody against CD31 (1:800). The slides were washed three times with PBS and then incubated for 1 hour at room temperature with goat anti-rat Cy-5 secondary antibody (1:500). Nuclear counterstain with Sytox green was applied for 10 minutes, and mounting medium (90% glycerol, 10% PBS, and 0.1 mol/L propyl gallate) was placed on each sample with a glass coverslip (Fisher Scientific, Pittsburgh, PA). Endothelial cells (CD31-positive cells) were identified by green fluorescence, whereas cells positive for EGFR, pEGFR, VEGFR2, pVEGFR2, PDGFR-β, or pPDGFR-β were identified by red fluorescence. The expression of EGFR, pEGFR, VEGFR2, pVEGFR2 PDGFR-β, or pPDGFR-β on endothelial cells was detected by co-localization of red and green fluorescence, which produced yellow staining. Frozen sections of cecal tumors were used for double immunofluorescence staining. Specimens were cut into 4-μm sections, mounted on positively charged slides, and stored at −80°C. Slides were fixed for 10 minutes in cold acetone, placed in a light-shielded humidified chamber, incubated for 20 minutes with protein blocking solution (5% normal horse serum and 1% normal goat serum in PBS) at room temperature, and incubated overnight at 4°C with goat anti-mouse IgG, Fab fragment (Jackson ImmunoResearch), to block endogenous immunoglobulins. The slides were again blocked briefly in protein blocking solution and incubated overnight at 4°C with primary mouse anti-EGFR (1:100) or mouse anti-VEGFR2 (1:400). The slides were washed three times with PBS and then incubated for 1 hour at room temperature with goat anti-mouse Cy-5 secondary antibody (1:500). Then, the slides were incubated overnight at 4°C with rabbit anti-VEGFR2 (1:400) or rabbit anti-PDGFR-β (1:400). The slides were washed with PBS for 3 minutes three times and then incubated for 1 hour at room temperature with goat anti-rabbit Cy-3 secondary antibody (1:500). Nuclear counterstain with Sytox green was applied for 10 minutes, and mounting medium was placed on each sample with a glass coverslip (Fisher Scientific). EGFR- or VEGFR2-positive cells were identified by green fluorescence, whereas VEGFR2- or PDGFR-β-positive cells were identified by red fluorescence. Co-localization of EGFR and PDGFR-β, VEGFR2 and PDGFR-β, or EGFR and VEGFR2 was detected by yellow staining. Confocal fluorescence images were collected using 20× or 40× objectives on a Zeiss LSM 510 laser scanning microscopy system (Carl Zeiss Inc., Thornwood, NY) equipped with a motorized Axiplan microscope, argon laser (458/477/488/514 nm, 30 mW), HeNe laser (413 nm, 1 mW, and 633 nm, 5 mW), LSM 510 control and image acquisition software, and appropriate filters (Chroma Technology Corp., Brattleboro, VT). Confocal images were exported to Adobe Photoshop software for preparation of montages. The intensity of cytoplasmic staining for TGF-α, EGF, VEGF, PDGF-B, EGFR, pEGFR, VEGFR2, pVEGFR2, PDGFR-β, and pPDGFR-β was evaluated in 10 random fields at 200× magnification and confirmed by an image analyzer using the Optimas image analysis software program (Bioscan, Edmonds, WA; Media Cybernetics, Silver Spring, MD). For negative controls, nonspecific IgG was used as the primary antibody. The staining intensity of the negative control was considered the background staining intensity and was measured and subtracted from the value of each sample. Intensity of staining for TGF-α, EGF, VEGF, PDGF-B, EGFR, pEGFR, VEGFR2, pVEGFR2, PDGFR-β, and pPDGFR-β was graded on a scale of 0 to 3+, with 0 representing no detectable staining and 3+ representing the strongest staining. The expression was defined as high if the score was 2+ or 3+ and low if the score was 0 or 1+. In the 12 surgical specimens of colon carcinomas that we examined, TGF-α, EGF, VEGF, and PDGF-B were mainly expressed on tumor cells, and expression of these ligands correlated with advanced stages of the disease (Figure 1A; Table 1).Table 1Expression of Ligands and Their Receptors in Surgical Specimens of Human Colon CancerCase no.TGF-αEGFVEGFPDGF-BEGFRpEGFRVEGFR2pVEGFRPDGFR-βpPDGFR-βTCECTCECTCECTCECTCECSCTCECSCDuke's B1212222231212013012220110202332100300231021121233321010014120221210200113112520322222030202201161110101002020020007002122002313021011Duke's C83133222212210220229221122222212013012Duke's D103333333333330330331111221212131202302212333332221212033022High-expression cases (%)58336758678358754210033750508303367 Open table in a new tab Surgical specimens were analyzed for expression of EGFR, pEGFR, VEGFR2, pVEGFR2, PDGFR-β, and pPDGFR-β. Receptors and phosphorylated receptors were labeled red, and endothelial cells (CD31-positive cells) were labeled green. The expression of tyrosine kinase receptors by tumor-associated endothelial cells was detected by co-localization, ie, yellow color. In all cases, EGFR and pEGFR were expressed by both tumor cells and endothelial cells; rates of expression varied by cell type. The percentage of cells expressing EGFR and pEGFR on tumor cells in stage D and stage B surgical specimens was 67% and 58%, respectively. On tumor-associated endothelial cells in stage D and stage B surgical specimens, it was 83% and 75%, respectively. VEGFR2 and pVEGFR2 were expressed on tumor cells in only a few neoplasms but were expressed on tumor-associated endothelial cells in all neoplasms. PDGFR-β and pPDGFR-β were not expressed on tumor cells in any neoplasm; these molecules were expressed only on stromal cells (red) and tumor-associated endothelial cells (yellow) (Figure 1, B and C; Table 1). In the next series of experiments, we determined whether tumor-associated endothelial cells expressed multiple protein tyrosine kinase receptors. Images captured in identical regions of specimens clearly demonstrated heterogeneity with respect to expression of EGFR, VEGFR2, and PDGFR-β on tumor-associated endothelial cells (Figure 2). Human KM12SM and HT29 colon cancer cells were implanted into the cecum of nude mice, and 6 weeks later tumors were resected and processed for immunohistochemical analysis. Both KM12SM and HT29 cells from orthotopic human colon carcinomas growing in nude mice expressed high levels of TGF-α, EGF, VEGF, and PDGF-B (Figure 3A). EGFR and VEGFR2 were highly expressed on both tumor cells (red) and tumor-associated endothelial cells (yellow), and these receptors were phosphorylated in both KM12SM cells (Figure 3B) and HT29 cells (Figure 3C). As in clinical specimen of human colon carcinomas, PDGFR-β and pPDGFR-β were not expressed on tumor cells but rather were expressed on stromal cells (red) and tumor-associated endothelial cells (yellow) (Figure 3, B and C). These data confirmed that the orthotopic colon cancer model reflected colon tumors in patients. To determine the extent of intratumoral heterogeneity for expression of EGFR, VEGFR2, and PDGFR-β in colon carcinomas, we performed double immunofluorescence staining for EGFR and PDGFR-β, EGFR and VEGFR2, and VEGFR2 and PDGFR-β in a surgical specimen of colon carcinoma (case 10, Duke's D) and in orthotopic KM12SM and HT29 carcinomas growing in the cecal wall of nude mice. EGFR and VEGFR2 were expressed on both tumor cells and tumor-associated endothelial cells, whereas PDGFR-β was expressed only on stromal cells, including tumor-associated endothelial cells (Figure 4). On tumor cells, expression of EGFR and VEGFR2 was not uniform; many tumor cells expressed both EGFR and VEGFR2, some cells expressed only EGFR, and other cells expressed only VEGFR2 (Figure 4). In this study, we examined the expression of TGF-α, EGF, VEGF, and PDGF-β and their respective receptors in 12 human colon cancer surgical specimens and in orthotopic tumors produced by two distinct human colon carcinoma cell lines. The present data clearly demonstrate inter- and intratumoral heterogeneity in expression of these growth factors and their receptors and confirmed that orthotopic colon cancer animal models reflect the clinical reality. Different proteins produced by tumor cells regulate the interaction between tumor cells and the organ microenvironment.39Fidler IJ Critical determinants of metastasis.Semin Cancer Biol. 2002; 12: 89-96Crossref PubMed Scopus (347) Google Scholar Many protein tyrosine kinase receptors on tumor cells and tumor-associated endothelial cells can be induced or up-regulated by ligands produced by tumor cells via autocrine and paracrine pathways.40Fidler IJ Angiogenic heterogeneity: regulation of neoplastic angiogenesis by the organ microenvironment.J Natl Cancer Inst. 2001; 93: 1040-1041Crossref PubMed Scopus (92) Google Scholar Treatment of cancer by inhibition of protein tyrosine kinase receptors has produced promising results.5Herbst RS Maddox AM Rothenberg ML Small EJ Rubin EH Baselga J Rojo F Hong WK Swaisland H Averbuch SD Ochs J LoRusso PM Selective oral epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 is generally well-tolerated and has activity in non-small-cell lung cancer and other solid tumors: results of a phase I trial.J Clin Oncol. 2002; 20: 3815-3825Crossref PubMed Scopus (548) Google Scholar, 6Mathew P Thall PF Jones D Perez C Bucana C Troncoso P Kim SJ Fidler IJ Logothetis C Platelet-derived growth factor receptor inhibitor imatinib mesylate and docetaxel: a modular phase I trial in androgen-independent prostate cancer.J Clin Oncol. 2004; 22: 3323-3329Crossref PubMed Scopus (111) Google Scholar, 7Strumberg D Richly H Hilger RA Schleucher N Korfee S Tewes M Faghih M Brendel E Voliotis D Haase CG Schwartz B Awada A Voigtmann R Scheulen ME Seeber S Phase I clinical and pharmacokinetic study of the novel Raf kinase and vascular endothelial growth factor receptor inhibitor BAY 43–9006 in patients with advanced refractory solid tumors.J Clin Oncol. 2005; 23: 965-972Crossref PubMed Scopus (787) Google Scholar However, in many clinical trials, the presence of targets in patients was not confirmed, and the response rate was unpredictable.41Thatcher N Chang A Parikh P Rodrigues Pereira J Ciuleanu T von Pawel J Thongprasert S Tan EH Pemberton K Archer V Carroll K Gefitinib plus best supportive care in previously treated patients with refractory advanced non-small-cell lung cancer: results from a randomised, placebo-controlled, multicentre study (Iressa Survival Evaluation in Lung Cancer).Lancet. 2005; 366: 1527-1537Abstract Full Text Full Text PDF PubMed Scopus (1986) Google Scholar For inhibition of phosphorylated EGFR, the presence of mutated receptors was reported to be a predictable variable,42Lynch TJ Bell DW Sordella R Gurubhagavatula S Okimoto RA Brannigan BW Harris PL Haserlat SM Supko JG Haluska FG Louis DN Christiani DC Settleman J Haber DA Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib.N Engl J Med. 2004; 350: 2129-2139Crossref PubMed Scopus (9823) Google Scholar but significant therapeutic responses of cancer cells with wild-type receptors has also been reported.4Yokoi K Sasaki T Bucana CD Fan D Baker CH Kitadai Y Kuwai T Abbruzzese JL Fidler IJ Simultaneous inhibition of EGFR, VEGFR, and platelet-derived growth factor receptor signaling combined with gemcitabine produces therapy of human pancreatic carcinoma and prolongs survival in an orthotopic nude mouse model.Cancer Res. 2005; 65: 10371-10380Crossref PubMed Scopus (89) Google Scholar, 43Busby JE Kim SJ Yazici S Nakamura T Kim JS He J Maya M Wang X Do KA Fan D Fidler IJ Therapy of multidrug resistant human prostate tumors in the prostate of nude mice by simultaneous targeting of the epidermal growth factor receptor and vascular endothelial growth factor receptor on tumor-associated endothelial cells.Prostate. 2006; 66: 1788-1798Crossref PubMed Scopus (23) Google Scholar These confusing criteria for selection of patients strongly indicate the need for better methodologies. The data reported here demonstrating inter- and intratumoral heterogeneity for expression of EGFR, VEGFR2, and PDGFR-β in different human colon cancer specimens of different stages suggest that targeting a single tyrosine kinase receptor is not likely to provide significant therapeutic results. In other words, targeted therapy is effective only against its target, and eliminating tumor cells that are dependent on one pathway, eg, EGFR, is not likely to prevent the proliferation of tumor cells that are independent of this pathway. Our results agree with a recent report demonstrating biological heterogeneity of tyrosine kinase receptors in other cancers.44Moroni M Veronese S Benvenuti S Marrapese G Sartore-Bianchi A Di Nicolantonio F Gambacorta M Siena S Bardelli A Gene copy number for epidermal growth factor receptor (EGFR) and clinical response to anti-EGFR treatment in colorectal cancer: a cohort study.Lancet Oncol. 2005; 6: 279-286Abstract Full Text Full Text PDF PubMed Scopus (886) Google Scholar, 45Ciardiello F Bianco R Caputo R Caputo R Damiano V Troiani T Melisi D De Vita F De Placido S Bianco AR Tortora G Antitumor activity of ZD6474, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, in human cancer cells with acquired resistance to anti-epidermal growth factor receptor therapy.Clin Cancer Res. 2004; 10: 784-793Crossref PubMed Scopus (320) Google Scholar These data suggest that more effective therapy for human colon cancers will require inhibition of multiple receptor pathways. This issue is not unique to colon cancer. We previously reported that simultaneous blocking of two protein tyrosine kinase pathways produced more efficient therapeutic effects in prostate3Kim SJ Uehara H Yazici S Langley RR He J Tsan R Fan D Killion JJ Fidler IJ Simultaneous blockade of platelet-derived growth factor-receptor and epidermal growth factor-receptor signaling and systemic administration of paclitaxel as therapy for human prostate cancer metastasis in bone of nude mice.Cancer Res. 2004; 64: 4201-4208Crossref PubMed Scopus (97) Google Scholar and pancreatic46Yokoi K Kim SJ Thaker P Yazici S Nam DH He J Sasaki T Chiao PJ Sclabas GM Abbruzzese JL Hamilton SR Fidler IJ Induction of apoptosis in tumor-associated endothelial cells and therapy of orthotopic human pancreatic carcinoma in nude mice.Neoplasia. 2005; 7: 696-704Abstract Full Text PDF PubMed Scopus (31) Google Scholar carcinoma than did blocking of a single such pathway. Therapeutic efficacy was further increased when three protein tyrosine kinase pathways were inhibited.4Yokoi K Sasaki T Bucana CD Fan D Baker CH Kitadai Y Kuwai T Abbruzzese JL Fidler IJ Simultaneous inhibition of EGFR, VEGFR, and platelet-derived growth factor receptor signaling combined with gemcitabine produces therapy of human pancreatic carcinoma and prolongs survival in an orthotopic nude mouse model.Cancer Res. 2005; 65: 10371-10380Crossref PubMed Scopus (89) Google Scholar Cancers are biologically heterogeneous for multiple properties, including antigenicity, sensitivity to chemotherapeutic agents, invasion, and metastasis.47Fidler IJ Critical factors in the biology of human cancer metastasis: twenty eighth G. H. A Clowes Memorial Award Lecture.Cancer Res. 1990; 50: 6130-6138PubMed Google Scholar, 48Fidler IJ Modulation of the organ microenvironment for the treatment of cancer metastasis.J Natl Cancer Inst. 1995; 87: 1588-1592Crossref PubMed Scopus (215) Google Scholar The present data demonstrate intratumoral heterogeneity in expression of growth factors and their receptors in colon cancers. The progressive growth of metastasis and survival of tumor cells depend on their interaction with the organ microenvironment. The expression of multiple tyrosine kinase receptors by different tumor cells within a single neoplasm indicates that targeting a single tyrosine kinase may not produce eradication of the disease. We thank Stephanie Deming for critical editorial review and Arminda Martinez for expert assistance with the preparation of the manuscript.
Referência(s)