After 5 days, cell numbers were measured using a tetrazolium salt-based assay (Figure). Optical densities are shown rather than normalized values to demonstrate the differential growth rates of the cell lines with respect to one another. All cell lines were growth-inhibited in a dose-dependent manner by the addition of Safinur. However, in the case of 786-O (renal cell adenocarcinoma), only the highest concentration was effective.

Cell Mobility Experiments. Using three variations of cell migration assays, we show that SafinurTM completely inhibits cell motility in all cell lines. 

Representative photographs are shown in the accompanying figure.  SafinurTM inhibits cell migration. Confluent monolayers were wounded vertically (standard wound assay, A and B). Panels A and B show the inhibition of normal fibroblasts and T-47D breast cancer cell migration by SafinurTM using the traditional monolayer wound assay. To better define the wound edge from the migrating cells, we modified this assay and increased the severity of the wound; this allowed the edge of the monolayer to curl, making it more visible. Using RPMI-7951 melanoma cells, we clearly show their migratory inhibition by SafinurTM (Fig. 3C). In panel D, we demonstrate a similar effect of SafinurTM on the radial migration of LN-18 glioblastoma cells. In all cases, detached cells were washed away and SafinurTM was added to 0.01% in fresh culture medium. Cells were fixed and stained on day 3. Dotted lines mark original edges. Magnification: 100· (A,B); 40· (C); and 20· (D).

(A) The migrating tumor cell spheroids on day 5, with the camera focusing on the cells. (B) The same field as (A) with the camera focusing on the filter. On day 10, filters were removed, fixed, and stained. (C) The stained filters at the conclusion of the experiment. Magnification: 100X. Using this highly invasive, spheroid-forming colon carcinoma cell line LoVo, we found that Safinur completely blocked the invasion of tumor cells through a thick, undiluted Matrigel layer to the other side of the filter membrane (Panel C). During this experiment, we noticed that Safinur-treated spheroids were disrupted and were incapable of moving through the matrix (Panels A and B). By focusing on the filter (Panel B), it is clear that the tumor spheroids in the Safinur-treated wells are far from the membrane whereas in the control, they appear close by.

Table.  Experimental outcomes and survival for control and SafinurTM-treated mice.

In order to obtain samples for analysis, we resected incompletely regressed tumors on day 5 of SafinurTM treatment (not shown). We noticed that the SafinurTM treated mice had tumors of a non-cohesive, crumbly consistency, unlike tumors from non- treated animals, which were more fibrous and solid. We attempted to develop primary tumor cell lines from these SafinurTM-treated tumors without success. Therefore, it was necessary to shorten the treatment to 2 days. Since we were unsure whether the mice would drink enough in a 2-day period, we injected SafinurTM intraperitoneally. After 2 days, the tumor was removed and analyzed (Fig. 2). This tumor appeared heterogeneous (Fig. 2A, inset) and was still vascularized. Parts of the tumor, especially those near blood vessels, exhibited a crumbly consistency (Fig. 2B) containing clusters of disrupted cells (Fig. 2C). The small fragments that separated from the main tumor mass during dissection were mostly composed of crumbly cell clusters (Fig. 2D) and resembled the disrupted tumors we observed in the in vitro assays (Figs. 4 and 5). Intriguingly, we also observed a crystalline accumulation in the crumbly parts of the tumor (Fig. 2D, arrowhead). These crystals were similar in shape and size to SafinurTM crystals in a saturated solution (not shown) but their identity remains undetermined.