The persistence of irradiated KS-IMM cells in vivo is short-term. (A) Nonirradiated and irradiated GFP⁺ KS-IMM cells (5 × 10⁶) were coinjected s.c. with MOLT-3 cells in a 1:1 ratio into NOD/SCID mice. The percentage of GFP⁺ KS-IMM cells in the tumor mass at different time points after the injection was measured by FACS analysis and is reported on the y axis; the percentage of CD5⁺ MOLT-3 cells was also determined and is indicated on the x axis. (Upper) Irradiated GFP⁺ KS-IMM cells disappeared rapidly from the injection site, and, 4 days later, they were virtually undetectable. (Lower) Control nonirradiated KS-IMM cells were detected at different time points after their coinjection with MOLT-3 cells, and their percentage increased with time. (B) Measurement by real-time PCR analysis of the amount of the GFP gene in cells recovered from the injection site at different time points. Results are expressed as the relative GFP content in each sample, assuming the undiluted DNA from the KS-IMM GFP⁺ cell line as 1. Detection of the GFP gene was rapidly lost after injection of irradiated KS-IMM cells, whereas it persisted in controls receiving nonirradiated KS-IMM cells. One representative experiment of two is shown.

Effects of angiogenic factors on MOLT-3 cell engraftment. (A) Effect of angiogenic factors on MOLT-3 tumorigenicity in NOD/SCID mice. MOLT-3 cells (5 × 10⁶ cells per injection, n = 6 mice per group) were injected in Matrigel either without (▿) or with (▾) bFGF (100 ng/ml), VEGF (•) (100 ng/ml), or irradiated KS-IMM cells (○). The tumor volume (mm³) was plotted as a function of time (weeks after transplantation). (B) Immunohistochemical analysis of 7-week-old dormant and progressively growing tumors. Shown is a representative CD31 staining of microvessels of dormant and progressively growing MOLT-3 tumors, induced by KS-IMM cell coadministration, recombinant VEGF, or bFGF. (Scale bars, 100 μm.) (C) Tumor growth curves of retroviral-vector-transduced MOLT-3 cells in NOD/SCID mice. Parental MOLT-3 cells or its derivatives, releasing either bFGF (▾) or VEGF (•) (5 × 10⁶ cells per injection, n = 6 mice per group), were injected either alone or in combination with irradiated MOLT-3-GFP⁺ (■), MOLT-3-bFGF (▿), or MOLT-3-VEGF (○) cells in a 1:1 ratio. The tumor volume was plotted as a function of time (weeks after transplantation). The tumor volumes of the experimental groups (▾), (•), (▿), and (○) evaluated 6 weeks after the beginning of the experiment significantly differed (P = 0.021) compared with the control groups. (D) Hematoxylin and eosin staining of tumors generated in the presence of sustained (MOLT-3bFGF) or short-term (MOLT-3 plus MOLT-3bFGF irr) bFGF release; one representative sample of three analyzed is shown. (Scale bars, 100 μm.)

Engraftment of MOLT-3 cells is fostered by coadministration of Kaposi’s sarcoma cells. (A) Macroscopic appearance of the tumors formed by MOLT-3 cells in NOD/SCID mice. (Left) Picture obtained 8 weeks after the bilateral s.c. injection of 5 × 10⁶ MOLT-3 cells. Although these cells failed to produce a visible mass in living mice, small tumors were found at autopsy in 11 of 50 sites injected with MOLT-3 cells (22%); tumor volumes ranged between 180 and 310 mm³. (Right) Picture obtained 8 weeks after bilateral s.c. injection of 5 × 10⁶ MOLT-3 cells plus 5 × 10⁶ irradiated KS-IMM cells; tumors were found in 56 of 56 injection sites (100%), and their volumes ranged between 1,240 and 1,780 mm³. (B) Flow-cytometric analysis of in vitro-cultured MOLT-3 and KS-IMM cells (in vitro) or single-cell suspensions obtained through mechanical dissociation of the tumors (ex vivo). Cells were incubated with a phycoerythrin-labeled anti-human CD5 mAb and analyzed on an EPICS-Elite cytofluorimeter. The percentage of positive cells is indicated. (C) Expression of angiogenic factors and cytokines in MOLT-3 and KS-IMM cells. PCR products were evaluated by electrophoresis on 1.5% agarose gels with ethidium bromide staining. (D) Tumor growth curves of s.c. xenotransplants in NOD/SCID mice. MOLT-3 cells (5 × 10⁶ cells per injection, n = 6 mice per group) were injected either alone (▾) or with other cell types. The tumor volume was plotted as a function of time (weeks after transplantation). Groups (•) and (■) received MOLT-3 cells inoculated with equal numbers of irradiated KS-IMM cells or irradiated MOLT-3 cells, respectively; group (○) received MOLT-3 cells into one flank and irradiated KS-IMM cells into the contralateral flank. In a group of mice (▿), irradiated KS-IMM cells were injected 21 days after MOLT-3 cell injection. The tumor volumes of the experimental group (•) evaluated after 9 weeks differed significantly (P = 0.021, according to Wilcoxon’s paired test) compared with the (▿) experimental group and the (▾), (○), and (■) groups.

Induction of angiogenesis by KS-IMM precedes tumor growth, and angiogenesis inhibitors counteract the engraftment of MOLT-3 cells. (A) Induction of angiogenesis in the s.c. Matrigel-pellet model by inclusion of cells in the pellet. The hemoglobin content per 100 mg of pellet is expressed as OD₅₄₀. The bars indicate mean ± SD; data were pooled from two independent experiments, each with six pellets per experimental group. The asterisks mark OD significantly different compared with control group (P = 0.021, according to Wilcoxon’s paired test). (B) Immunohistochemical analysis of vascularization of Matrigel pellets isolated from MOLT-3-injected and MOLT-3 plus KS-IMM-injected NOD/SCID mice 6 days after injection. Anti-CD31 staining showed microvessels penetrating the Matrigel sponges only in MOLT-3 plus KS-IMM samples. (C) Angiogenesis inhibitors counteract KS-mediated engraftment of MOLT-3 cells. KS-IMM cells were transduced by retroviral vectors producing either murine angiostatin or murine IFNα. These cells were then irradiated and coinjected s.c. with MOLT-3 cells in a 1:1 ratio into NOD/SCID mice. Tumor growth curves of xenotransplants (5 × 10⁶ MOLT-3 cells, n = 6 mice per group) of MOLT-3 cells in combination with nontransduced (•), angiostatin-producing (○) or IFNα-producing (▾) KS-IMM cells are shown. Angiostatin significantly delayed (P = 0.021), and IFNα completely prevented (P = 0.021), the generation of progressively growing MOLT-3 tumors.