Published OnlineFirst February 26, 2014; DOI: 10.1158/1541-7786.MCR-13-0532-T Molecular Cancer Genomics Research Spontaneous Reversion of the Angiogenic Phenotype to a Nonangiogenic and Dormant State in Human Tumors Michael S. Rogers1,3,4, Katherine Novak1,3, David Zurakowski2, Lorna M. Cryan1,3,4, Anna Blois1,3,7, Eugene Lifshits1,3, Trond H. Bø, Anne M. Oyan5, Elise R. Bender1,3, Michael Lampa1,3, Soo-Young Kang1,3, Kamila Naxerova4, Karl-Henning Kalland5,6, Oddbjorn Straume7,8, Lars A. Akslen7, Randolph S. Watnick1,3,4, Judah Folkman†1,3,4, and George N. Naumov1,3,4 Abstract The angiogenic switch, a rate-limiting step in tumor progression, has already occurred by the time most human tumors are detectable. However, despite significant study of the mechanisms controlling this switch, the kinetics and reversibility of the process have not been explored. The stability of the angiogenic phenotype was examined using an established human liposarcoma xenograft model. Nonangiogenic cells inoculated into immunocompro- mised mice formed microscopic tumors that remained dormant for approximately 125 days (vs. <40 days for angiogenic cells) whereupon the vast majority (>95%) initiated angiogenic growth with second-order kinetics. These original, clonally derived angiogenic tumor cells were passaged through four in vivo cycles. At each cycle, a new set of single-cell clones was established from the most angiogenic clone and characterized for in vivo for tumorigenic activity. A total of 132 single-cell clones were tested in the second, third, and fourth in vivo passage. Strikingly, at each passage, a portion of the single-cell clones formed microscopic, dormant tumors. Following dormancy, like the original cell line, these revertant tumors spontaneously switched to the angiogenic phenotype. Finally, revertant clones were transcriptionally profiled and their angiogenic output determined. Collectively, these data demonstrate that the angiogenic phenotype in tumors is malleable and can spontaneously revert to the nonangiogenic phenotype in a population of human tumor cells. Implications: Leveraging the rate of reversion to the nonangiogenic phenotype and tumor dormancy may be a novel anticancer strategy. Mol Cancer Res; 12(5); 754–64. Ó2014 AACR. Introduction thus cease macroscopic growth—is rare (2). Nevertheless, In cancer, a tumor's switch to angiogenesis is a rate- there are a limited number of reports of tumors that cease limiting step in its progression from microscopic to macro- macroscopic growth, suggesting that such a reversion of the scopic size (1). As a result, small, occult tumors are a common angiogenic switch may occur (3, 4). finding on autopsy of individuals who die of nonneoplastic Regulation of angiogenesis is typically viewed as a switch causes (primary studies summarized in ref. 2). In contrast, the whose state is governed by the relative local concentration of converse finding—of tumors that switch off angiogenesis and angiogenesis stimulators and inhibitors. The switch meta- phor is common in the field because of the strong biphasic nature of angiogenesis. In experimental models of pathologic conditions, it is rare to observe small, stepwise accumulation Authors' Affiliations: Departments of 1Surgery and 2Anesthesia; 3the Vascular Biology Program, Boston Children's Hospital; 4Harvard Medical of additional vessels. Rather it is common to observe that School, Boston, Massachusetts; 5Department of Microbiology, Haukeland once angiogenesis has been initiated, vessel growth proceeds University Hospital; 6Section for Microbiology, The Gade Institute; 7Centre for Cancer Biomarkers CCBIO, Department of Clinical Medicine; and throughout the pathologic process. In addition, variation in 8Section of Oncology, Institute of Internal Medicine, University of Bergen, intensity of angiogenesis has been observed, and seems to be Bergen, Norway a major rate-limiting factor in tumor growth. For example, Note: Supplementary data for this article are available at Molecular Cancer substantial variation in microvessel density is observed Research Online (http://mcr.aacrjournals.org/). throughout tumors, with regions exhibiting the highest † Died January 14, 2008. density predicting the overall growth rate of a tumor, metastatic status, and patient survival (5–7). Corresponding Author: Michael S. Rogers, Boston Children's Hospital, 11.211 Karp Family Research Bldg., 300 Longwood Avenue, Boston, MA In contrast with the tumor as a whole, we and others have 02115. Phone: 617-919-2252; Fax: 617-730-0231; E-mail: shown that individual tumor cells can exhibit significant [email protected] variation in their ability to induce angiogenesis. For example, doi: 10.1158/1541-7786.MCR-13-0532-T when individual cell clones derived from a primary human Ó2014 American Association for Cancer Research. liporsarcoma are implanted in immunocompromised mice, 754 Mol Cancer Res; 12(5) May 2014 Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst February 26, 2014; DOI: 10.1158/1541-7786.MCR-13-0532-T Reversion of Angiogenesis the timing of the transition to macroscopic growth varies the second, third, and fourth in vivo passage, respectively. In from 7 to >160 days (8). In addition, several commercially vitro growth curves were generated using a Coulter counter. available tumor cell lines, originally derived from human tumors that were macroscopic in size, exhibit extended Subcutaneous tumor growth periods of preangiogenic growth, ranging from a few weeks, Male SCID mice, 6 to 8 weeks of age (MGH), were cared to years, to the lifespan of the animal (9). However, an for in accordance with the standards of the Institutional experimentally induced increase in the angiogenic output of Animal Care and Use Committee, and used under an these tumors (e.g., by transfection with VEGF; refs. 10, 11) approved protocol. Tumors were generated by injecting 5 Â results in early and rapid macroscopic growth. Similarly, 106 cells in 0.2 mL PBS subcutaneously into the shaved alterations occurring during extended evolution of dormant lower right flank. Injection sites were palpated weekly for tumors in mice can result in increased net angiogenic output. tumors (typically detected at 50 mm3) and measured every In at least one system, this was accompanied by a decrease in 3 to 4 days thereafter, with volumes calculated using the expression of angiogenic inhibitors (e.g., thrombospondin- formula 0.52W2L. Mice were euthanized when tumor 1), rather than an increase in angiogenic stimulators (e.g., volume reached approximately 1,000 mm3 or mice showed VEGF; ref. 12). Importantly, in all of these experiments in signs of discomfort. vitro growth rates for the angiogenic sublines (which are derived from nonangiogenic parental lines) did not differ Immunohistochemistry and histology significantly from the growth rate of nonangiogenic sublines. Excised tumors were rinsed in ice-cold PBS and fixed in These findings exclude differences in cell division rate as a 4% paraformaldehyde. Paraffin-embedded tissue was sec- mechanism for the observed differences in macroscopic tioned (4 mm) and representative sections (5/tumor) were growth. stained with H&E, proliferating cell nuclear antigen (PCNA; Finally, experiments in which angiogenic cells were PC10; 1:150; DAKO), and CD31 (PECAM; 1:250; BD admixed with nonangiogenic cells before inoculation in mice Biosciences). have demonstrated that even a minority of proangiogenic cells is sufficient to induce growth (and metastasis) in the entire Quantitation of angiogenesis regulators tumor (11). Nontransformed (i.e., stromal) cells have also Serum-free medium (15 mL) was incubated on day-old been shown to play a critical role in the induction of cultures (5 Â 106 cells/15-cm plate) for a further 24 hours. angiogenesis in some tumors (13). These observations lead Concentrations of human VEGF165 (R&D), bFGF (R&D), to the notion that the angiogenic switch may be an ensemble and Tsp-1 (CYT Immune Science) were measured by ELISA property comprised of contributions from all the cells in the kits using the manufacturers' protocols with control values tumor, rather than an obligate property of only the tumor- (serum-free media) subtracted and results normalized per igenically transformed cells in a tumor (10). Therefore, it is 104 cells. Protein lysates from angiogenic and revertant cell possible that individual tumor cells, although derived from an clones were western blotted as previously described (13) for angiogenic tumor, may not possess the angiogenesis-inducing Tsp-1 (Ab11; LabVision), and b-actin (Abcam), using potential of that tumor. We sought to test that possibility by horseradish peroxidase–conjugated goat anti-mouse (Jack- serial in vivo passage, cloning, and quantitative analysis of the son Immunoresearch Labs) for detection. growth phenotypes of such individual tumor cells. In addi- tion to assessing the stability of the switch to an angiogenic Gene expression analysis phenotype in individual cells, these experiments allowed us to Independent angiogenic (two) and nonangiogenic assess the nature of the events that lead to the angiogenic (three) cell lines from the second cycle of cloning were switch, and determine that it is not comprised of a single step. analyzed using the Agilent Human Whole Genome (4 Â 44k) Oligo Microarray (Agilent Technologies Inc.) as fi Materials and Methods previously described (15). Signi cance analysis of micro- arrays was performed in J-Express