A strategy to eradicate well-developed Krebs-2 ascites in mice

We describe the strategy, which allows curing experimental mice engrafted with Krebs-2 ascites. The strategy is based on the facts that i) Krebs-2 tumor-initiating stem cells (TISCs) are naturally capable of internalizing fragments of extracellular double-stranded DNA (dsDNA); ii) upon delivery into TISCs, these dsDNA fragments interfere with the on-going DNA repair process so that TISCs either die or lose their tumorigenic potential. The following 3-step regimen of therapeutic procedures leading to eradication of Krebs-2 ascites is considered. Firstly, three timed injections of cyclophosphamide (CP) exactly matching the interstrand cross-link (ICL) repair phases that lead to synchronization of ascites cells in late S/G2/M. Secondly, additional treatment of ascites 18 hours post each CP injection (at NER/HR transition timepoint) with a composite dsDNA-based preparation interfering with the NER and HR repair pathways, so that tumorigenic properties of ascites cells are compromised. Thirdly, final treatment of mice with a combination of CP and dsDNA injections as ascites cells undergo apoptotic destruction, and the surviving TAMRA+ TISCs arrested in late S/G2/M phases massively enter into G1/S, when they regain sensitivity to CP+dsDNA treatment. Thus, this regimen assures that no viable cells, particularly Krebs-2 TISCs, remain.


SUPPLEMENTARY DATA Histopathology analysis of organs and tissues from ascites-engrafted mice subjected to treatments with CP or CP+dsDNA vs control mice
The present Annex features the results of histopathology analysis of organs and tissues isolated from agonizing mice and control animals. In total, the analysis was performed for two control group animals (No 1 and No 2), three CP group animals, two of which succumbed to the secondary ascites (No 1 and No 2) and the third died because of multiple organ failure (No 3), and one animal from the CP+dsDNA group that did not have obvious ascites but apparently died due to multiple organ failure.

Lung tissue
Lung tissue appeared essentially normal, yet it displayed somewhat reduced airness, thickened (swollen) interalveolar walls and small clusters of lymphocytes around the vessels (rarely) ( Figure 1A). In the second control animal, these features are much more pronounced, and lymphocytes infiltrate the interalveolar walls over a greater area ( Figure 1B).

Lymph nodes
In one animal, large tumorous nest was found sitting on the surface of the lymph node, which was separated by a thin sheath of connective tissue (Figure 2A). In the second animal, cancer cells were also found on the lymph  node serosa and in the sinus lumen ( Figure 2B). Several cancer cells were scattered across the paracortical zone.

Small intestine
Large cancer cell node was found on the intestinal serosa in one of the animals ( Figure 3A). The rest of the intestinal cell wall layers, submucous layer, as well as mucosa were normal ( Figure 3B).
In the second animal, tumor cells have almost entirely covered the small intestines outer wall ( Figure 3C).
The cancer nodule displayed small necrotic foci, with cancer cells spreading into the muscular wall ( Figure 3D). Intestinal mucosa remained essentially intact.

Large intestine
The situation is very similar to that described for the small intestine. The degree of invasion in the second animal is much higher. Even though the integrity of the mesenteric wall is not compromised, cancer cells are found under the external longitudinal layer (Figure 4).

Pancreas
In both animals, we observed that large cancer nodules were present on the surface of the pancreatic capsule. In the second animals, cells of the large cancer nodule were also found to invade the subcapsular space ( Figure 5).

Kidneys
No pathologies.

Adrenal glands
No pathologies.

Liver
The first animal in the control group had cancer cells located on the surface of the visceral peritoneum covering the liver ( Figure 6A). It invaded under the capsule at several sites ( Figure 6B) but never spread beyond the marginal layer. In the second animal, we observed a large cancer nodule with a central necrotic zone ( Figure 6C).  Primary tumor localization could not be unambiguously established, but it definitely was not growing from the surface, i.e. it is likely a metastasis.

Spleen
In both control animals, cancer nodules with or without necrotic zone were found on the splenic capsule (Figure 7).

Cancer nodules
Cancer nodules composed of the aggregated cancer cells were found. Most of them were found attached to the parietal peritoneum lining the abdominal wall. Cancer cells invaded the peritoneum and spread between the muscle fibers of abdominal wall.

CP-treated group Lungs
In both animals from this group, besides moderately swollen interalveolar walls, we observed clusters of cancer cells, some of which were destroyed (at least four such clusters are visible in the Figure 8A). At a higher magnification ( Figure 8B), one can see that these cancer cells are covered with endothelial cells and apparently myocytes. In other words, these structures likely correspond to small lymphatic vessels or to postcapillary venules.
In the animal No 3, lung parenchyma appears consolidated due to interstitial swelling and atelectasis; profuse lymphocytic infiltration and large necrotic zones are visible ( Figure 8C). Single cancer cells are frequent in the alveolar openings ( Figure 8D).

Small intestine
Scattered solitary cancer cells were present on the surface of the small intestine covered with peritoneum.

Large intestine
Normal.

Pancreas
In one animal, pancreatic tissue was heavily swallen and acini appeared necrotic. We attribute this to the obstruction in the pancreatic duct ( Figure 9).

Adrenal glands
In one animal from the CP group, the retroperitoneal cover of adrenal gland displayed solitary cancer cells (Figure 10), which may indicate that the tumor has disseminated through the parietal peritoneum close to the kidney.

Cancer nodules
Cancer nodules composed of the aggregated cancer cells (frequently with large regions of necrosis) were found ( Figure 11). Most of the nodules were attached to the parietal peritoneum. Cancer cells invaded the peritoneum and spread throughout the muscular layer of the abdominal wall.

CP+dsDNA group Lung
The only CP+dsDNA group animal studied had an intact lung tissue.

Small intestine
No pathology.

Large intestine
No pathology.

Spleen
No pathology.