Therapeutic approach of human peritoneal carcinomatosis with Dbait in combination with capnoperitoneum: proof of concept

Abstract

Background: Peritoneal carcinomatosis is an unmet medical need. Laparoscopy offers a unique opportunity to control and to steer the operating environment during surgery by loading carbon dioxide with a therapeutic substance and creating the so-called therapeutic capnoperitoneum. We have treated a human sample of peritoneal carcinomatosis from an endometrial adenocarcinoma ex vivo just after surgery.

Methods: A nontoxic therapeutic agent (Dbait) was aerosolized into a box containing diseased human peritoneum under a pressure of 12 mmHg CO(2). Dbait (noncoding DNA fragments) acts through jamming DNA damage sensing and signaling, ultimately inhibiting DNA repair system of cancer cells. Dbait were coupled to cholesterol molecules to facilitate intracellular uptake, and to Cyanine (Cy5) to allow detection by fluorescence. In a control experiment, the same solution was applied to the other half of the sample using conventional lavage.

Results: Physical results revealed fluorescence within the tumor up to 1 mm depth in the therapeutic capnoperitoneum sample and no uptake in the lavage sample. Biological results showed intranuclear phosphorylation of H2AX in the nebulized sample and no activity in the lavage sample. Importantly, tumor nodules showed more activity than the neighbor, normal peritoneum. Detection of histone gamma-H2AX (phosphorylated H2AX) reveals activation of DNA-dependent protein kinase (DNA-PK) by Dbait, which has been shown to be the key step for sensitization to genotoxic therapy.

Conclusions: Dbait are taken up by cancer cells and have a biological activity up to 1 mm depth. Nebulization of the molecule is significantly more effective than conventional lavage. This proof of principle supports the need for clinical studies applying therapeutic capnoperitoneum together with Dbait for treating peritoneal carcinomatosis.

Fig. 1

Laparoscopy-like ex vivo experiment on fresh operation specimen of diseased human peritoneum (peritoneal carcinomatosis from endometrial origin). Therapeutic capnoperitoneum (12 mmHg) was established in a plastic box. An electrostatic gradient of 60 V was generated between the MIP and the biological tissue, placed on a neutral electrode. MIP micropump

Fig. 2

Diffusion of therapeutic substance onto the peritoneum. I. Cryosection of human peritoneum, red: Dbait-Cy5 staining, blue: Dapi (nucleus). A After nebulization of Dbait, B after lavage with Dbait, C after nebulization without Dbait. Staining reveals homogeneous peritoneal fluorescence in the therapeutic capnoperitoneum sample (arrows), only minimal uptake in the lavage sample, and no staining in the control sample. II: H&E staining

Fig. 3

Penetration of therapeutic substance into the peritoneum. Cryosection of human peritoneum, red: Dbait-Cy5 staining, blue dapi (nucleus). A After nebulization of Dbait, B after lavage with Dbait, C after nebulization without Dbait. Staining reveals fluorescence up to seven cellular layers in the therapeutic capnoperitoneum sample (A), no tissue uptake in the lavage sample (B) and no staining in the control sample (C)

Fig. 4

Biological activity. Cryosection of human peritoneum. Detection of early Dbait activity marker: Phosphorylated H2AX. Red: Dbait-Cy5 staining, blue: Dapi staining (nucleus), green: γ-H2AX. A After nebulization of Dbait, B after lavage with Dbait. Intranuclear phosphorylation of H2AX is observed the nebulized sample and only artifactual staining (no nuclear staining) in the lavage sample. Detection of histone γ-H2AX (phosphorylated H2AX) reveals activation of DNA-Phosphokinase by Dbait, which has been shown to be the key step for sensibilization to genotoxic therapy

Fig. 5

Tumor specificity. Detection of early Dbait activity marker: cryosection of human peritoneum; A blue: Dapi staining (nucleus); B green: γ-H2AX after nebulization of Dbait, C red: Dbait-Cy5 staining, D merged: H2AX activation is maximal at the tumor invasion front (arrow)