Novel immunocompetent murine models representing advanced local and metastatic pancreatic cancer

Abstract

Background: The development of novel therapeutics for pancreatic cancer has been hindered by a lack of relevant preclinical models. The purpose of this study was to evaluate the clinical relevancy of two pancreatic cancer models using standard-of-care therapeutic agent gemcitabine.

Materials and methods: Murine Panc02 cells were injected directly into the spleen or pancreas of C57BL/6 mice to respectively create models of metastatic and locally advanced pancreatic cancer. Beginning 7 d post-Panc02 injection, treated mice received 20 mg/kg gemcitabine i.p. every 3 d. Animals were sacrificed when the untreated mice became moribund and tumor/liver weight used to assess tumor burden.

Results: Untreated mice became moribund 22 d after pancreatic Panc02 injection. Gross analysis revealed localized pancreatic tumors weighing 1.063 g. Intrasplenic Panc02 injection produced extensive liver metastasis by d 15 when the untreated mice first became moribund. Liver weights at this time averaged 3.6 g compared with the average non-tumor-bearing weight of 1.23 g. Gemcitabine therapy resulted in a 54% decrease in localized pancreatic tumor weight and 62.5% decrease in metastatic liver weight. Additionally, gemcitabine therapy extended animal survival to 20.5 d compared with 18.0 d average for the untreated mice.

Conclusions: We describe two models depicting both locally advanced and metastatic pancreatic cancer in immunocompetent mice. In efforts to establish baseline therapeutic efficacy, we determined that gemcitabine reduces tumor burden in both models and enhances survival in the metastatic model. These clinically relevant models provide valuable tools to evaluate novel therapeutics in pancreatic cancer.

Figure 1. Pancreatic and Intrasplenic Panc02-luc injections result in consistent tumor formation

A) Representative images and H&E sections of pancreatic tumors 22 days after tumor cell injection. Scale marks 1cm. B) Representative images and H&E sections of metastatic liver tumors 15 days after intrasplenic tumor cell injection.

Figure 2. Gemcitibine decreases tumor burden of localized and metastatic tumors and enhances survival

Pancreatic injection (A&B): Mice either received no treatment (n=5) or an i.p. injection of 20mg/kg gemcitabine on days 7, 10, 13, 16, and 19 post-tumor cell injection (n=9). All animals were sacrificed on day 22, their tumors isolated and tumor weight (A) and volume (B) taken as a measure of tumor burden. The group means are graphed along with the SEM. Intrasplenic Injection (C&D): C) mice either received no treatment (n=6) or an ip injection of 20mg/kg gemcitabine on days 7, 10, and 13 post-tumor cell injection (n=6). All animals were sacrificed on day 15 and their livers weighed as a measure of tumor burden. The group means are graphed along with the SEM. D) Mice were either left untreated (n=5) or treated with 20mg/kg gemcitabine on days 7, 10, 13, and 16 (n=6). Morbundity was determined by a blind investigator and plotted with a Kaplan Meier survival curve. *P<0.05 and **P<0.005

Figure 3. Gemcitabine decreases proliferation in the localized tumor model

Mice were treated with 20mg/kg gemcitabine on Days 7, 10, 13, 16, and 19 post-orthotopic Panc02 injection. Following sacrifice on Day 22, tumor sections were analyzed for apoptosis and proliferation. While gemcitabine therapy did not alter the number of TUNEL-positive cells (A, P>0.05), treatment did reduce the number of Ki-67 positive cells compared to the untreated group (B, **P<0.005). The group means of at least 3 random fields per sample are graphed along with the SEM.

Figure 4. Gemcitabine does not affect TUNEL or Ki-67 expression in metastatic liver tumors

Mice were treated with 20mg/kg gemcitabine on Days 7, 10, and 13 post-orthotopic Panc02 injection. Following sacrifice on Day 15, tumor sections were analyzed for apoptosis and proliferation using both random fields throughout the tumor (A) and on the tumor-border interface (B). There were no significant differences between untreated and gemcitabine-treated tumors with either TUNEL (left) or Ki-67 (right) quantification (P>0.05). The group means of at least 3 random fields per sample are graphed along with the SEM.

Figure 5. Bioluminescence Imaging does not accurately predict tumor burden

(A&B): Mice were received an intrasplenic injection of 1 million Panc02-luc cells and either received no treatment (n=6) or an i.p. injection of 20mg/kg gemcitabine on days 7, 10, and 13 post-tumor cell injection (n=6). On days 7 and 14, BLI was performed on all mice following an i.p. injection of 3mg D-luciferin. A) There were no statistically significant differences in BLI between the untreated and gemcitabine-treated groups on either day (P>0.05). B) Liver weight did not closely correlate with BLI output though linear regression showed a relationship did exist. (C) 1 million Panc02-luc cells were injected intrasplenically on day 0 (n=9). On day 16, mice were injected with 3mg D-luciferin and liver harvested for ex vivo BLI. Though a relationship exists (P<0.05), BLI output does not closely correlate with liver weight (R²=0.4727). (D) 1 million Panc02-luc cells were injected orthotopically on day 0 (n=9). On day 15, mice were injected with 3mg D-luciferin and pancreas tumors harvested for ex vivo BLI. BLI output does not correlate with tumor weight (P>0.05, R²=0.0920).