Effects of HyaRegen gel on tumour proliferation of colorectal peritoneal metastases

Abstract

Pressurized intraperitoneal aerosol chemotherapy (PIPAC) is a valuable therapeutic alternative for patients with peritoneal metastases. PIPAC uses a hyaluronic acid-based gel to reduce surgically induced adhesions. The aim of this study was to evaluate the effects of the hyaluronic acid-based gel on tumor dissemination. First, we explored whether the survival of CT26 luciferase-expressing murine colonic tumor cells was correlated with the dose of HyaRegen® Gel, and we determined the half-maximal inhibitory concentration (the IC50) of the gel. Next, we performed an in vitro study of cell survival rates after gel application on day 0 (D0) and day 1 (D1). Finally, we intraperitoneally administered the gel to mice with immunocompetent BALB/c colonic peritoneal metastases (on D0, D5, D10, D14, and D18). Tumor growth was regularly monitored using a bioluminescence assay (on D11, D17, and D21). After all mice had been sacrificed on D21, the body weights and the volumes of intraperitoneal ascites were measured; the Peritoneal Carcinosis Index (PCI) and Ki-antigen 67 scores were calculated. The IC50 value was 70 μL of gel in a total volume of 100 μL. The cell survival rates on D4 were identical in the control group and the two groups that had been treated with gel on D0 and D1. The bioluminescence levels over time were similar in the gel and control groups. The PCI scores were 35.5 ± 2.89 for the control group and 36 ± 2.45 for the gel group (p = 0.8005). The mean Ki-67 index percentages were 37.28 ±1 1.75 for the control group and 34.03 ± 8.62 for the gel group (p = 0.1971). This in vitro and in vivo study using a mouse model of immunocompetent metastatic peritoneal cancer did not reveal any pro- or anti-tumoral effect of HyaRegen® Gel. These findings indicate that the gel can be used to treat PIPACs with minimal apprehension.

Fig 1. CT26- luc cell viability is affected by increasing amount of gel on D4 after seeding.

A photograph of the cell cultures on D4 according to gel percentages (A). The percentages of viable cells on D4. Mean +-SD. * for p value <0.05, ** <0.01, *** <0.001 and **** <0.0001. (B). Logarithmic regression curve between gel concentration and viability indicates an IC50 around 7% of HayRegen® gel (C).

Fig 2. In vivo analysis of HyaRegen® Gel effects on tumor proliferation, dissemination and ascites.

In vivo quantification of tumor growth via bioluminescence tracking of luciferase-expressing CT26 cells on days 11, 17, and 21 after gel administration: BLI mice photography (A) and tumor growth histograms, mean +-SD,* for p value <0.05 (B). Comparison of ascites volume between the control and gel groups on D21, mean +-SD (C). Comparison of median peritoneal cancer index (PCI) between the control and gel groups (D).

Fig 3

Histograms showing in vivo proliferating cells (Ki-67 antigen labeling) for the control and gel groups, mean +-SD (A). Micrography showing Ki-67 (B) and HES staining (C).