Title: Abnormalities in Pericytes on Blood Vessels and Endothelial Sprouts in Tumors
Abstract: Endothelial cells of tumor vessels have well-documented alterations, but it is less clear whether pericytes on these vessels are abnormal or even absent. Here we report that α-smooth muscle actin (α-SMA) and desmin-immunoreactive pericytes were present on >97% of blood vessels viewed by confocal microscopy in 100-μm-thick sections of three different spontaneous or implanted tumors in mice. However, the cells had multiple abnormalities. Unlike pericytes on capillaries in normal pancreatic islets, which had desmin but not α-SMA immunoreactivity, pericytes on capillary-size vessels in insulinomas in RIP-Tag2 transgenic mice expressed both desmin and α-SMA. Furthermore, pericytes in RIP-Tag2 tumors, as well as those in MCa-IV breast carcinomas and Lewis lung carcinomas, had an abnormally loose association with endothelial cells and extended cytoplasmic processes deep into the tumor tissue. α-SMA-positive pericytes also covered 73% of endothelial sprouts in RIP-Tag2 tumors and 92% of sprouts in the other tumors. Indeed, pericyte sleeves were significantly longer than the CD31-immunoreactive endothelial cell sprouts themselves in all three types of tumors. All three tumors also contained α-SMA-positive myofibroblasts that resembled pericytes but were not associated with blood vessels. We conclude that pericytes are present on most tumor vessels but have multiple abnormalities, including altered expression of marker proteins. In contrast to some previous studies, the almost ubiquitous presence of pericytes on tumor vessels found in the present study may be attributed to our use of both desmin and α-SMA as markers and 100-μm-thick tissue sections. The association of pericytes with endothelial sprouts raises the possibility of an involvement in sprout growth or retraction in tumors. Endothelial cells of tumor vessels have well-documented alterations, but it is less clear whether pericytes on these vessels are abnormal or even absent. Here we report that α-smooth muscle actin (α-SMA) and desmin-immunoreactive pericytes were present on >97% of blood vessels viewed by confocal microscopy in 100-μm-thick sections of three different spontaneous or implanted tumors in mice. However, the cells had multiple abnormalities. Unlike pericytes on capillaries in normal pancreatic islets, which had desmin but not α-SMA immunoreactivity, pericytes on capillary-size vessels in insulinomas in RIP-Tag2 transgenic mice expressed both desmin and α-SMA. Furthermore, pericytes in RIP-Tag2 tumors, as well as those in MCa-IV breast carcinomas and Lewis lung carcinomas, had an abnormally loose association with endothelial cells and extended cytoplasmic processes deep into the tumor tissue. α-SMA-positive pericytes also covered 73% of endothelial sprouts in RIP-Tag2 tumors and 92% of sprouts in the other tumors. Indeed, pericyte sleeves were significantly longer than the CD31-immunoreactive endothelial cell sprouts themselves in all three types of tumors. All three tumors also contained α-SMA-positive myofibroblasts that resembled pericytes but were not associated with blood vessels. We conclude that pericytes are present on most tumor vessels but have multiple abnormalities, including altered expression of marker proteins. In contrast to some previous studies, the almost ubiquitous presence of pericytes on tumor vessels found in the present study may be attributed to our use of both desmin and α-SMA as markers and 100-μm-thick tissue sections. The association of pericytes with endothelial sprouts raises the possibility of an involvement in sprout growth or retraction in tumors. Blood vessels in tumors are recognized as a clinically important therapeutic target.1Jain RK The next frontier of molecular medicine: delivery of therapeutics.Nat Med. 1998; 4: 655-657Crossref PubMed Scopus (350) Google Scholar The abnormalities of tumor vessels provide the potential for targeting these vessels without destroying the normal vasculature.2Arap W Pasqualini R Ruoslahti E Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model.Science. 1998; 279: 377-380Crossref PubMed Scopus (1846) Google Scholar, 3Pasqualini R Vascular targeting with phage peptide libraries.Q J Nucl Med. 1999; 43: 159-162PubMed Google Scholar Tumor vessels are recognized as dynamic, both in terms of the formation of new vessels by angiogenesis and the remodeling of existing vessels.4Folkman J Tumor angiogenesis.Adv Cancer Res. 1985; 43: 175-203Crossref PubMed Scopus (1188) Google Scholar, 5Gilead A Neeman M Dynamic remodeling of the vascular bed precedes tumor growth: MLS ovarian carcinoma spheroids implanted in nude mice.Neoplasia. 1999; 1: 226-230Abstract Full Text PDF PubMed Scopus (55) Google Scholar Tumor vessels also express novel molecules that can serve as selective targets for therapeutic agents,6Pasqualini R Koivunen E Kain R Lahdenranta J Sakamoto M Stryhn A Ashmun RA Shapiro LH Arap W Ruoslahti E Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis.Cancer Res. 2000; 60: 722-727PubMed Google Scholar, 7St. Croix B Rago C Velculescu V Traverso G Romans KE Montgomery E Lal A Riggins GJ Lengauer C Vogelstein B Kinzler KW Genes expressed in human tumor endothelium.Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1632) Google Scholar and have structural and functional abnormalities, such as leakiness, that are important for the accessibility of drugs to cancer cells or other cellular constituents of tumors.8Yuan F Dellian M Fukumura D Leunig M Berk DA Torchilin VP Jain RK Vascular permeability in a human tumor xenograft: molecular size dependence and cutoff size.Cancer Res. 1995; 55: 3752-3756PubMed Google Scholar, 9Hobbs SK Monsky WL Yuan F Roberts WG Griffith L Torchilin VP Jain RK Regulation of transport pathways in tumor vessels: role of tumor type and microenvironment.Proc Natl Acad Sci USA. 1998; 95: 4607-4612Crossref PubMed Scopus (1994) Google Scholar, 10Hashizume H Baluk P Morikawa S McLean JW Thurston G Roberge S Jain RK McDonald DM Openings between defective endothelial cells explain tumor vessel leakiness.Am J Pathol. 2000; 156: 1363-1380Abstract Full Text Full Text PDF PubMed Scopus (1297) Google ScholarWith the increasing promise of vascular targeting in cancer, a thorough understanding of the cellular structure and function of tumor vessels becomes even more important, as this information is key to interpreting the effects of anti-angiogenic agents. 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Pericytes on normal capillaries typically express desmin but not α-SMA, whereas normal venular pericytes express both molecules.26Nehls V Drenckhahn D The versatility of microvascular pericytes: from mesenchyme to smooth muscle?.Histochemistry. 1993; 99: 1-12Crossref PubMed Scopus (191) Google Scholar Marker expression can also vary in different organs and with pathological conditions.18Schlingemann RO Rietveld FJ de Waal RM Ferrone S Ruiter DJ Expression of the high molecular weight melanoma-associated antigen by pericytes during angiogenesis in tumors and in healing wounds.Am J Pathol. 1990; 136: 1393-1405PubMed Google Scholar, 34Sundberg 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, 35Buschard K Horn T Aaen K Josefsen K Persson H Fredman P Presence of sulphatide (3′-sulphogalactosylceramide) in pericytes in the choroid layer of the eye: sharing of this glycolipid autoantigen with islets of Langerhans.Diabetologia. 1996; 39: 658-666Crossref PubMed Scopus (18) Google Scholar, 37Schlingemann RO Oosterwijk E Wesseling P Rietveld FJ Ruiter DJ Aminopeptidase A is a constituent of activated pericytes in angiogenesis.J Pathol. 1996; 179: 436-442Crossref PubMed Scopus (69) Google Scholar Because no single commonly used marker identifies all pericytes with certainty, there may be problems with identifying pericytes in pathological conditions such as cancer when the cells change their expression of marker proteins.18Schlingemann RO Rietveld FJ de Waal RM Ferrone S Ruiter DJ Expression of the high molecular weight melanoma-associated antigen by pericytes during angiogenesis in tumors and in healing wounds.Am J Pathol. 1990; 136: 1393-1405PubMed Google Scholar, 34Sundberg 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, 37Schlingemann RO Oosterwijk E Wesseling P Rietveld FJ Ruiter DJ Aminopeptidase A is a constituent of activated pericytes in angiogenesis.J Pathol. 1996; 179: 436-442Crossref PubMed Scopus (69) Google ScholarLittle is known about the variability of marker expression by pericytes in tumors because most studies have used a single marker, usually α-SMA or desmin, and have equated lack of immunoreactivity with lack of pericytes.21Benjamin LE Golijanin D Itin A Pode D Keshet E Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal.J Clin Invest. 1999; 103: 159-165Crossref PubMed Scopus (1044) Google Scholar, 22Eberhard A Kahlert S Goede V Hemmerlein B Plate KH Augustin HG Heterogeneity of angiogenesis and blood vessel maturation in human tumors: implications for antiangiogenic tumor therapies.Cancer Res. 2000; 60: 1388-1393PubMed Google Scholar, 38Rangdaeng S Truong LD Comparative immunohistochemical staining for desmin and muscle-specific actin. 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Birkhauser Verlag, Basel1997: 29-46Crossref Scopus (11) Google Scholar Some of these differences may be explained by differences in pericyte marker expression among tumors. However, others are likely to result from differences in the markers used to identify pericytes or differences in section thickness, where partial pericyte coverage was missed in thin histological sections.One reason for determining whether pericytes are a consistent feature of tumor vessels is to explore whether they participate in angiogenesis by guiding newly formed blood vessels through their association with endothelial sprouts.41Nehls V Denzer K Drenckhahn D Pericyte involvement in capillary sprouting during angiogenesis in situ.Cell Tissue Res. 1992; 270: 469-474Crossref PubMed Scopus (326) Google Scholar, 42Tsuzuki H Sasa S Ultrastructural observation of capillary sprouts in the dental organs of rat molars.Kaibogaku Zasshi. 1994; 69: 684-696PubMed Google Scholar Another reason is to examine the suggestion that the absence of pericytes sensitizes tumor vessels to withdrawal of vascular endothelial growth factor (VEGF).21Benjamin LE Golijanin D Itin A Pode D Keshet E Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal.J Clin Invest. 1999; 103: 159-165Crossref PubMed Scopus (1044) Google ScholarIn the present study, we compared the amount of pericyte coverage of tumor vessels, as determined by immunoreactivity of two markers, α-SMA and desmin, with that of normal vessels. We also questioned whether the relationship of pericytes to endothelial cells in tumors differs from that in normal tissues and examined the relationship of pericytes to endothelial sprouts. We compared three tumors in mice: spontaneous pancreatic tumors in RIP-Tag2 mice, implanted MCa-IV mouse mammary carcinomas, and Lewis lung carcinomas. Pericytes and endothelial cells were co-localized in immunohistochemically stained 100-μm-thick sections and examined by confocal microscopy. A preliminary description of this work has been reported.43Morikawa S Baluk P Jain RK McDonald DM Pericytes closely associate with endothelial cells of vascular sprouts in mouse tumors.Proc Am Assoc Cancer Res. 2000; 41: 87Google ScholarMaterials and MethodsAnimals and Preparation of TumorsSpontaneous pancreatic islet cell tumors were studied in RIP-Tag2 transgenic mice with a C57BL/6 background. In these mice, expression of the SV40 virus T antigen is driven by the rat insulin promoter.44Hanahan D Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes.Nature. 1985; 315: 115-122Crossref PubMed Scopus (1006) Google Scholar Mice expressing the viral oncogene were identified by genotyping tail-tip DNA by the polymerase chain reaction, and tumors were studied when the mice reached 10 weeks of age.44Hanahan D Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes.Nature. 1985; 315: 115-122Crossref PubMed Scopus (1006) Google Scholar Implanted MCa-IV mouse mammary carcinomas9Hobbs SK Monsky WL Yuan F Roberts WG Griffith L Torchilin VP Jain RK Regulation of transport pathways in tumor vessels: role of tumor type and microenvironment.Proc Natl Acad Sci USA. 1998; 95: 4607-4612Crossref PubMed Scopus (1994) Google Scholar and Lewis lung carcinomas (American Type Culture Collection, Rockville, MD) were studied in syngeneic male C3H and C57BL/6 mice, respectively (25 to 30 g body weight).10Hashizume H Baluk P Morikawa S McLean JW Thurston G Roberge S Jain RK McDonald DM Openings between defective endothelial cells explain tumor vessel leakiness.Am J Pathol. 2000; 156: 1363-1380Abstract Full Text Full Text PDF PubMed Scopus (1297) Google Scholar Two-mm cubes of tumor were implanted under the dorsal skin, and the tumors were examined 10 to 20 days later when they had reached a diameter of 8 to 12 mm. Mice were housed under barrier conditions in the animal care facility at University of California at San Francisco. All of the experimental procedures were approved by the University of California at San Francisco Committee on Animal Research.Lectin Staining and Perfusion Fixation of VasculatureMice were anesthetized with ketamine (87 mg/kg) plus xylazine (13 mg/kg) injected intramuscularly. In some animals, fluorescein isothiocyanate (FITC)-labeled Lycopersicon esculentum lectin (100 μg in 100 μl of 0.9% NaCl; Vector Laboratories, Burlingame, CA) was injected into the femoral vein and allowed to circulate for 3 minutes before perfusion of fixative.10Hashizume H Baluk P Morikawa S McLean JW Thurston G Roberge S Jain RK McDonald DM Openings between defective endothelial cells explain tumor vessel leakiness.Am J Pathol. 2000; 156: 1363-1380Abstract Full Text Full Text PDF PubMed Scopus (1297) Google Scholar The chest was opened rapidly, and the vasculature was perfused for 3 minutes at a pressure of 120 mmHg with fixative [4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline (PBS), pH 7.4] from a 18-gauge cannula inserted into the aorta via an incision in the left ventricle.10Hashizume H Baluk P Morikawa S McLean JW Thurston G Roberge S Jain RK McDonald DM Openings between defective endothelial cells explain tumor vessel leakiness.Am J Pathol. 2000; 156: 1363-1380Abstract Full Text Full Text PDF PubMed Scopus (1297) Google Scholar The fixative was not preceded by a saline rinse. The right atrium was incised to create a route for the fixative to escape. After removal, tissues were stored in fixative at 4°C until they were processed for immunohistochemistry.Immunohistochemistry and ImagingSpecimens were rinsed several times with PBS, embedded in 10% agarose, and cut with a Vibratome or infiltrated overnight with 30% sucrose, frozen, and cut with a cryostat. Sections 100 μm in thickness were incubated at room temperature for 12 to 15 hours in a mixture of anti-mouse CD31 (PECAM-1, clone MEC 13.3 rat monoclonal, 1:500; Pharmingen, San Diego, CA) antibody for endothelial cell identification plus anti-α-SMA (Cy3-conjugated mouse monoclonal, clone 1A4, 1:1000; Sigma Chemical Co., St. Louis, MO) or anti-desmin (rabbit polyclonal 1:2000; DAKO Corp., Carpinteria, CA) antibody for pericyte identification. Antibodies were diluted with PBS containing 0.01% thimerosal as an anti-bacterial and 0.3% Triton X-100 to improve penetration of 100-μm sections. After several rinses with PBS, specimens were incubated for 6 hours at room temperature with a goat anti-rat or goat anti-rabbit secondary antibody labeled with FITC or Cy5 for CD31 staining or with Cy3 for desmin staining (antibodies from Jackson Immuno Research, West Grove, PA). After mounting in Vectashield (Vector Laboratories, Burlingame, CA), specimens were examined with a Zeiss Axiophot fluorescence microscope and a Zeiss LSM 410 laser-scanning confocal microscope. Confocal images were stored as digital files, viewed with Photoshop (Adobe, Mountain View, CA), and printed on a Fujix Pictography 3000 color printer (Fuji Photo Film Inc., Elmsford, NY).Transmission Electron MicroscopyTumors, fixed by vascular perfusion of 0.5% glutaraldehyde and 1% paraformaldehyde in 0.075 mol/L sodium cacodylate buffer, pH 7.4, were removed, immersed in 2.5% glutaraldehyde in cacodylate buffer for a minimum of 2 hours, and embedded in 10% agarose.10Hashizume H Baluk P Morikawa S McLean JW Thurston G Roberge S Jain RK McDonald DM Openings between defective endothelial cells explain tumor vessel leakiness.Am J Pathol. 2000; 156: 1363-1380Abstract Full Text Full Text PDF PubMed Scopus (1297) Google Scholar Sections 100 μm in thickness were cut with a Vibratome. Specimens measuring ∼1 × 3 mm were cut from the sections, treated with OsO4 and uranyl acetate, dehydrated, and embedded in epoxy resin.45McDonald DM Endothelial gaps and permeability of venules in rat tracheas exposed to inflammatory stimuli.Am J Physiol. 1994; 266: L61-L83PubMed Google Scholar Sections 0.5 μm in thickness were stained with toluidine blue for light microscopy, and sections 50 to 100 nm in thickness were stained with lead citrate and examined with a Zeiss EM-10 electron microscope.Morphometric MeasurementsMorphometric measurements of blood vessels were made on images obtained from 100-μm-thick sections of four specimens (n = 4 mice) from each of the three tumors and normal tissues, unless designated otherwise. Regions of necrosis were avoided. The sections were double-stained for CD31 and α-SMA immunoreactivities. Real-time video images were viewed or digital images were captured with a Zeiss Axiophot microscope equipped with single and dual filters for FITC and Cy3 and a low-light, three-chip CoolCam CCD camera (SciMeasure Analytical Systems, Atlanta, GA). Measurements were made using image analysis software developed for this purpose in our laboratory.45McDonald DM Endothelial gaps and permeability of venules in rat tracheas exposed to inflammatory stimuli.Am J Physiol. 1994; 266: L61-L83PubMed Google Scholar The proportion of vessels covered by pericytes was determined for 50 vessels in each specimen. Pericytes were considered present if α-SMA or desmin immunoreactivity was visible anywhere around the vessel perimeter. The number, length, and pericyte coverage of endothelial sprouts were determined in the same specimens. Sprouts were identified as tapered CD31-immunoreactive processes that extended away from the main axis of a vessel and ended abruptly. For all of the sprouts identified on vessels in each specimen (10 to 27 sprouts were analyzed per 50 vessels in each specimen), the lengths of the CD31-immunoreactive endothelial cell strands (sprout length) and the surrounding α-SMA immunoreactive pericyte sleeve (pericyte sleeve length) were measured. In addition, for 10 sprouts from each specimen that had FITC-lectin staining in vivo (n = 2 mice for each type of tumor), the lengths of the lectin-stained lumen (lumen length) and the associated CD31-immunoreactive endothelial cell strands were measured. The extent of pericyte coverage on vessels was determined on 15 properly cross-sectioned vessels in each specimen by measuring the proportion of CD31-positive vessel perimeter covered by α-SMA-immunoreactive cells. Values are expressed as means ± SEM (SEM). The significance of differences between means was assessed by analysis of variance followed by the Bonferroni/Dunn test (P < 0.05).Resultsα-SMA and Desmin Immunoreactivity of PericytesNormal PericytesIn normal pancreatic acini and islets, α-SMA-immunoreactive cells were abundant on arterioles and venules but not on capillaries (Figure 1A). By comparison, desmin immunoreactive cells were p