Single cell derived clones were generated using a limiting dilution method from the parental U-87 MG human glioblastoma cell line. Thirteen clones were chosen according to similar rapid kinetics of colony formation in tissue culture wells. RNA was extracted from each clone and the relative expression level of Thrombospondin (TSP-1), a well-known endogenous inhibitor of angiogenesis that has been shown to be elevated in all dormant tumors 
, was determined using real time PCR (). When compared with the expression level of the parental U-87 MG cell line, most (10 out of 13) of the clones had lower TSP-1 expression, while only 3 clones (#1, #2 and #6) had elevated TSP levels. Clone #1 had a significant increase in TSP level (over 25-fold higher expression than in parental U-87 MG cell line).
Gene expression analysis of single cell-derived clones from U-87 MG glioblastoma cell line.
Since a high TSP level could suggest slow kinetics of tumor growth, we chose to focus our analysis on three clones with varying TSP levels: Clone #1, with the highest TSP level, Clone #6 with an intermediate TSP level, and Clone #7 with a very low TSP level (marked by arrows in ). We postulated that Clone #1 might generate dormant tumors, whereas clones #6 and #7 would generate intermediate or fast-growing tumors, respectively. We then continued to analyze the expression levels of several tumor dormancy-associated genes we had previously identified 
. First, we tested the expression levels in the three clones of the additional genes that were previously shown to be upregulated in dormant tumors (). Clearly, angiomotin (Amot) and IGFBP5 levels were upregulated only in Clone #1. Notably, IGFBP5 expression was around 1000-fold higher than in the parental U-87 MG cell line. Expression of TGF-β2 was upregulated in all clones tested.
Genes previously shown to be elevated in fast-growing tumors were expected to be observed as downregulated in tumor cells that form dormant or slow-growing tumors. Such downregulation was indeed observed in Clone #1 for CD73, EGFR, and most significantly for ESM-1 (), strengthening our prediction that this clone could generate dormant tumors.
Tumor growth patterns were then analyzed in SCID mice. Equal numbers of cells were injected subcutaneously (s.c.) from each clone and from the parental U-87 MG cell line, and tumor growth was monitored (). As expected, the parental U-87 MG cells generated very small tumors (volume below 100 mm3), which after 3–4 weeks initiated rapid growth. Similar ‘bi-phasic’ growth kinetics were observed for tumors generated from clones #6 and #7. Although Clone #6, which had an intermediate level of TSP, initially formed tumors larger than the parental cell line, its tumors grew slower in the rapid growth phase. Clone #7, which had a very low level of TSP, formed tumors smaller than those generated by the parental cell line or by Clone #6. Importantly, Clone #1 formed dormant tumors which remained indolent and were barely detectable by gross examination throughout the experiment (). This confirmed our hypothesis that the parental U-87 MG cell line contains cells which when isolated will form dormant tumors, and that Clone #1 was generated from such cells.
Comparison of tumor growth patterns and characteristics.
At the end point of the experiment, tumors generated by clones #6 and #7 were clearly smaller than those generated by the parental U-87 MG cells. Although smaller in mass, Clone #6 and Clone #7 tumors were highly vascularized and tightly capsulated, similar to tumors generated from parental U-87 MG cells (). In contrast, tumors generated from Clone #1 could be detected only after flipping the skin and seemed avascular. These tumors were occasionally found attached to the muscle tissue instead of the skin, like most tumors from U-87 MG, Clone #6, and Clone #7 ().
The fate of indolent tumors generated by Clone #1 was analyzed by following their tumor growth over a prolonged period of time lasting more than 200 days (Fig. S1
). As expected, while U-87 MG tumors grew rapidly in the first 3–4 weeks after inoculation, tumors from Clone #1 remained undetectable for over 70 days. Three of the four tumors from Clone #1 eventually emerged from dormancy and initiated growth at 81, 122, and 127 days post inoculation (Fig. S1
). One mouse injected with Clone #1 cells never developed any detectable tumors during the 270 days of the experiment (data not shown). Tumors that originated from Clone #1 cells remained at the site of injection in a constant small size without expanding in mass for a long period of time (i.e., dormant). Importantly, once these tumors emerged from dormancy and started growing, the growth rate could be as rapid as in the parental U-87 MG cell line derived tumors.
To evaluate tumor properties in their orthotopic microenvironment in a non-invasive manner, both Clone #1 and U-87 MG parental cells were infected with mCherry as previously described 
. Then, in order to assure that the infection did not alter tumor characteristics, SCID mice were inoculated s.c. with either cell line, and dormancy periods were monitored and compared. Tumors generated by cells from Clone #1 remained dormant and avascular for more than 70 days, while tumors generated from the parental U-87 MG cell line were highly vascularized and palpable 20 days following inoculation (). Following the escape from dormancy, mCherry-labeled Clone #1 tumors showed a similar tumor growth rate pattern to mCherry-labeled U-87 MG tumors ().
Tumor growth patterns of mCherry-labeled tumor cells.
Cellvizio® imaging of the vasculature of U-87 MG tumors revealed enlarged, highly tangled, and non-continuous vessels with wider lumen and blunt ends, leakage, and sluggish blood flow. These are typical signs of the enhanced permeability and retention (EPR) effect phenomenon for macromolecules such as Dextran-FITC at 70 kDa size used here (). In contrast, blood vessels in Clone #1 tumors at a size of 2 mm3 are almost non-appearing. For comparison, normal vessels in healthy tissues adjacent to these tumors are shown to have continuous blood flow and a regular shape with anastomosis.
To compare the angiogenic potential, in vivo
analysis of size-matched tumors (~2 mm3
) from U-87 MG and Clone #1 was performed (). The presence of the tumors at the site of injection was validated by the mCherry fluorescent signal and later by H&E staining (). U-87 MG tumors were significantly more vascularized compared with size-matched tumors generated from Clone #1 cells, as observed by gross examination of the tumors after flipping the skin, by analysis of CD34 positively-stained cells and by presence of blood vessels in H&E staining (marked with arrows) in the U-87 tumor sections ( and Fig. S2
). Microbubbles contrast-enhanced ultrasound (US) imaging not only supported these results, but also emphasized the functionality of the blood vessels by extensive blood flow within U-87 MG tumors, as opposed to no detectable blood flow within Clone #1 tumors (). These results are in accordance with high TSP-1 expression in tumors generated by Clone #1 compared to none in U-87 MG tumors (). Interestingly, Clone #1 tumors express TSP-1 exclusively prior to the escape from dormancy, when tumor size is approximately 2 mm3
(). Tumors generated from Clone #1 that had emerged from dormancy and reached a diameter of over 1500 mm3
do not express TSP-1. U-87 MG tumors do not express TSP-1 in any stage of tumor progression.
Comparison of size-matched tumors generated from U-87 MG and Clone #1 cells.
With the purpose of investigating the growth rate of orthotopic tumors, mCherry-labeled U-87 MG and Clone #1 cells were inoculated by stereotactic injections to the striatum of SCID mice. Tumor growth patterns were monitored and compared (). Whereas tumors originating from Clone #1 cells remained at a small size for more than 30 days, tumors originating from the parental U-87 MG cells initiated growth and mass expansion 8 days post inoculation. The dormant phenotype of tumors generated by Clone #1 does not depend, therefore, on the site of injection, but is rather an intrinsic property of the tumor cells.
Orthotopic tumor growth patterns.
We next compared the in vitro proliferation kinetics, migration, invasiveness and cell morphology of U-87 MG and Clone #1 cells. No significant changes were observed in cell structure and morphology in culture (). Cell growth kinetics for 72 hours were similar when U-87 MG cells were compared with Clone #1 cells (). Furthermore, no difference was found between the migration ability of U-87 MG and Clone #1 cells (). However, the ability of the cancer cells to migrate and invade through the endothelial cell monolayer differs significantly. The migration of U-87 MG cells through endothelial cells towards serum-free media was significantly higher compared with that of Clone #1 cells (). These observations suggest that the dormancy seen in tumors generated from Clone #1 cells does not result from slower cell proliferation or migration, but could relate to their relative lack in invasion capacity as demonstrated in the transendothelial assay.
In vitro cellular characteristics of U-87 MG and Clone #1 cells.
The angiogenic potential of the tumor cells was evaluated by comparing the effect of conditioned media (C.M.) collected from U-87 MG and Clone #1 cells on endothelial cells in a series of assays which represent different steps in the angiogenesis process (). Differences in the angiogenic potential of the two cell lines were demonstrated by extensive proliferation and sprouting following 7 days of incubation in the presence of C.M. from U-87 MG cells, compared with negligible sprouting in the presence of C.M. from Clone #1 cells (). Also, HUVEC exhibited significantly higher migration rates towards C.M. from U-87 MG cells, compared with that towards C.M. from Clone #1 cells (). Interestingly, HUVEC migrated toward C.M. from U-87 MG at a significantly faster rate than toward medium supplemented with 10% serum (positive control) (). Similarly, increased numbers of tube-like structures and vascular network areas were observed in the presence of U-87 MG C.M. compared with the number found in HUVEC in the presence of both C.M. from Clone #1 cells or medium supplemented with 10% serum ().
Angiogenic potential comparison between cells of U-87 MG and Clone #1.
HUVEC’s ability to form capillary-like structures in the presence of C.M. from Clone #1 cells was enhanced compared with that in the presence of medium supplemented with 10% serum, indicating the presence of factors that positively affect HUVEC’s ability to form tube-like structures (). The functionality of the vasculature recruited by factors secreted by the cancer cells was assessed using 3D contrast-enhanced ultra-sound analysis of s.c. Matrigel® plugs containing C.M. from the two cell lines (). The vascularization within plugs containing C.M. from U-87 MG cells was significantly higher, when compared to vascularization within plugs containing C.M. from Clone #1 cells. Clearly, the level of pro-angiogenic factors that positively affect endothelial cells’ ability to proliferate, sprout, migrate, form tube-like structures and finally form functional blood vessels is higher within C.M. from U-87 MG cells as compared to C.M. from Clone #1 cells.
These observations suggest that U-87 MG cells are more angiogenic than Clone #1 cells. Since the angiogenic potential of cells depends on the ratio of pro- and anti-angiogenic factors, it is possible that Clone #1 overexpresses factors which negatively affect angiogenesis. In order to assess the balance between pro- and anti-angiogenic factors (either directly or indirectly affecting endothelial cells) produced by the cancer cells, an equal total number of cells (1×106
) were injected s.c. into SCID mice as mixed populations of U-87 MG and Clone #1 in different ratios: 1
10, and 1
100, respectively. Tumor progression was followed and compared to the single population (). When 50% and 90% of cells implanted were of Clone #1, delayed escape from dormancy was observed by approximately 10 and 20 days, respectively. When the cell population consisted of 99% Clone #1 and only 1% U-87 MG cells, a dormancy period identical to that of the Clone #1 single-population was observed. This suggests the presence of anti-angiogenic factors, overexpressed mainly by Clone #1 cells.
Tumor growth patterns of mixed cancer cell populations.