Existence of a small subset of cancer cells referred to as tumor initiating stem cells (TISCs) largely responsible for tumor progression and resistance to chemotherapeutic cytostatic drugs reperesent an important recent paradigm shift. The present work is the first report in the series of papers from our group where we describe the development of anticancer therapy based on the selective targeting of TISCs. Here were characterize a cytoreductive activity of cyclophosphamide (CP), double-stranded DNA (dsDNA) and combinations thereof against the TISC population present in mouse Krebs-2 ascites. We evaluated engraftment potential of Krebs-2 cancer cells treated in ascites-bearing mice in vivo, followed by re-engraftment to congenic recipient mice in a form of a solid graft. These data indicate that with our approach TISCs can be completely eliminated even from a well-established ascites. We demonstrate that dsDNA-internalizing and CD34-positive cells are more sensitive to the synergistic effects of CP and dsDNA. When Krebs-2 ascites are treated with human DNA 1-12 hours post CP injection, this results in either elimination of cells that internalize TAMRA-labeled DNA (TISCs) or alters their phenotype, which is accompanied with the loss of surface expression of CD34. Next, we show that the timepoint 18 hrs post CP treatment is critical to the ongoing repair process in that it divides the repair into two phases: nucleotide excision repair + dsDNA break repair and homologous recombination. Importantly, both of these phases can be conveniently used for targeting the tumorigenic potential of the graft. In the context of monotherapy, CP is most effective against ascites grafts when administered as serial injections. To achieve maximum efficiency, the timing of consecutive injections must match the time when cancer cells found at G2/M during the first injection enter G1/S and/or the time of active repair via homologous recombination.

In the present paper, we report on the series of experiments where multiple regimens of CP and dsDNA injections were tested for targeting the ascites form of murine Krebs-2 cancer in situ. We show that combining CP with cross-linked human and salmon dsDNA results in a synergistic toxicity for ascites-bearing mice, an observation supported by the histopathology analysis of organs and tissues of experimental animals. In contrast, using a composite mixture of native and cross-linked human and salmon DNA after CP injections leads to a significant increase in average lifespan of the treated mice. Further, we demonstrate that repeated rounds of CP+dsDNA injections result in dramatic anticancer effect. The timing of injections is chosen so that they target the cells that were insensitive to the previous treatments as they were in the G2/M phase. 3-4 rounds of injections are needed to eliminate the subpopulation of tumor-initiating cancer stem cells. Our experiments identified the regimen when complete resorption of the primary Krebs-2 ascites occurs in all of the treated animals, followed by a remarkable remission period lasting 7-9 days. Yet, this regimen does not prevent secondary site metastases (either solid or ascites form) from developing, which is likely caused by the migration of ascites cells into adjacent tissues or by incomplete eradication of cancer stem cells. To address these and other questions, we expanded the study and performed histopathology analysis, which indicated that secondary metastases is not the only cause of death. In fact, many animals displayed unfolding systemic inflammatory reaction which was culminated by multiple organ failure. Thus, we developed the concept for treating ascites form of Krebs-2 cancer, which allows elimination of the primary ascites.