Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The goal of this report was to provide a detailed protocol for performing ultrasound-guided implantation of KPC-derived PDA cell lines. In the representative experiments shown in Figure 2-4, we confirm that UG-OTIM tumors grow at a consistent rate and in a dose-dependent manner. Furthermore, we show that UG-OTIM tumors recapitulate the salient immunological and histological features of the KPC TME. Thus, the UG-OTIM system is a preclinical PDA mouse model that can be used in a high-throughput manner to rapidly screen new treatment combinations in vivo.

Using the UG-OTIM protocol outlined here, the mice were prepared for implantation, secured to the heated ultrasound platform and the positions of both the platform and the transducer were adjusted for this procedure as shown in Figure 1A. High-resolution ultrasound imaging was used to identify an injection site within the mouse pancreas that could be targeted without perforation of either the kidney or spleen (Figure 1B). Under ultrasonographic visualization, the needle was carefully introduced to the abdominal cavity through the peritoneal wall and guided into the mouse pancreas (Figure 1C). After correct placement of the needle was established, the tumor cell suspension was injected very slowly into the pancreas. A successful implantation was confirmed by the presence of a bubble within the pancreas (Figure 1D). In early experiments, the mouse was sacrificed, and efficacy of the procedure was verified by directly visualizing a fluid bolus in the gross pancreas tissue (Figure 1E).

Tumor implantation and growth rate was monitored by weekly ultrasound imaging. Successful implantations produced tumors that were contained within the borders of the pancreas throughout the time of the experiment (Figure 2A). The ultrasound software (see Table of Materials) used allowed for tumor area and volume to be determined for each time point as well as for 3D mapping of measured tumors to be generated (Figure 2B), and the 3D images were confirmed at the time of mouse necropsy (Figure 2C). An improper cell injection during the UG-OTIM procedure can result in the development of a peritoneal wall tumor (a representative image of which is shown in Figure 2D). Mice that present with peritoneal tumors can be excluded from further studies.

To determine the concentration of cells optimal for use in preclinical studies, a C57Bl/6 KPC-derived PDA cell line (4662)⁹ was injected in to naïve, wild-type C57Bl/6 mice across six independent experiments. This cell line (passaged in vitro six times) was derived from a fully backcrossed KPC tumor-bearing mouse (>10 generations, confirmed by SNP-analysis¹⁹) to prevent molecular histocompatibility complex mismatch tumor rejection antigens. Tumor cells were injected at a low titer (1.25 x 10⁶ cells/25µL) and at a high titer (5 x 10⁶ cells/25µL) dose. The high titer tumor injections resulted in a larger proportion of tumor-bearing animals three weeks after injections compared to the low titer cohort (Figure 3A). Despite the delay in tumor onset, the overall tumor growth rate was not significantly different between the two doses (Figure 3B). Similarly, while the survival rate between the two cohorts was not significantly different, the data trend towards slightly improved survival in the low titer cohort (Figure 3C). The high titer cohort also produced a greater proportion of mice with tumors that were enrollable in preclinical studies (designated at ≥20mm³ tumor volume) by four weeks post-injection than the low titer cohort (Figure 3D). The majority of mice from both high and low titer cohorts presented with enrollable tumors by day 25 and developed end-stage disease symptoms including ascites (data not shown). Metastases, which do not occur using 4662 at the cell doses from this protocol, may be modeled with different cell lines or doses³³.

The UG-OTIM method required a mastery of fine motor skills to accurately localize the desired injection site, which was challenging in our earliest experiments. For this reason, we included a table depicting the number of animals that developed tumors within the pancreas (successful implantations) compared to the total number of animals that underwent ultrasound-guided tumor implantation (Table 1). Animals were eliminated from future analyses if tumors developed in an undesired location (i.e., kidney) or if there was no evidence of tumor by six weeks post-injection, as indicated. The weekly progression of the proportion of mice bearing enrollable pancreatic tumors (≥20mm³ tumor volume) from each experiment is also shown in Table 1.

To determine if there was a benefit to using the UG-OTIM approach rather than the traditional surgical orthotopic model (beyond time investment after gaining proficiency at the technique), we compared the seeding of PDA tumors in the peritoneal wall of mice after each procedure. We found that only 2/31 mice (6.5%) developed unintended peritoneal wall tumors after UG-OTIM injections, as compared 7/15 mice that developed peritoneal wall tumors after surgical injection (46.6%, p < 0.0029) (Table 1). Thus, the rate of seeding additional tumors in the peritoneum is greatly reduced in the UG-OTIM method as compared to surgical implantation.

Upon sacrifice of mice bearing UG-OTIM tumors, we found that the gross anatomy of the tumors was similar to spontaneous KPC tumors (Figure 4A). Histological analysis demonstrated a pattern of abnormal ductal structures that was similar in both models and that recapitulated the morphology of the human disease (Figure 4B). To investigate the immune infiltrate within both KPC and UG-OTIM tumors, representative histological samples were stained for CD3 (expressed by T cells) and F4/80 (expressed by macrophages). In both models, staining patterns revealed tumors that were poorly infiltrated by T cells (Figure 4C), but highly infiltrated by macrophages (Figure 4D). This finding is consistent with the immunologically cold phenotype of most human and KPC PDA samples^5,7,12.

Figure 1: Ultrasound-guided implantation of PDA cells into the murine pancreas. (A) Orientation of mouse, ultrasound stage, and ultrasound probe used to obtain a high-resolution image of abdominal organs. Note the stage and platform have been turned 90° from the standard orientation to allow easy access to the upper left quadrant of mouse abdomen. (B) Ultrasonographic image depicting the identification of kidney, spleen and pancreas. Here the injection needle is positioned against the mouse abdomen. (C) Ultrasonographic image depicting the needle within the mouse pancreas. (D) Ultrasonographic image depicting the bubble at the injection site (outlined in blue) following controlled injection of tumor cells in to the pancreas. (E) Laparotomy revealing the fluid bolus containing PDA cells in the pancreas after ultrasound guided injection. Please click here to view a larger version of this figure.

Figure 2: Monitoring tumor growth after UG-OTIM injection. (A) Representative ultrasonographic image of UG-OTIM tumor (outlined in blue) at 2, 3, and 5 weeks post-injection, as indicated. (B) Representative reconstructed 3D image of UG-OTIM tumor 5 weeks post-injection using the ultrasound software. (C) Representative gross anatomy of UG-OTIM tumor 5 weeks post-injection upon mouse necropsy. (D) Representative ultrasonographic image of a peritoneal wall tumor growing in the subcutaneous layer after improper cell injection at 7 weeks post-injection. Please click here to view a larger version of this figure.

Figure 3: Dose-dependent onset of enrollable tumors following ultrasound guided injection of PDA cells. (A) Proportion of tumor-bearing mice at indicated time points after injection as described in Figure 1 with high titer (500,000 cells/25µL) or low titer (125,000 cells/25µL) tumor cells, as indicated. (B) Tumor growth kinetics of mice from (A), each symbol represents a group of mice, error bars indicate SEM. (C) Proportion of mice from (A) alive at indicated time points after injection. Mice were euthanized or censored if tumors were >1000mm³ or body condition was poor due to tumor comorbidities. (D) Time to enrollable tumor onset for mice from (A). Enrollable tumors are considered to be ≥20mm³ in volume. Data representative of 4 independent experiments with n=8-20 mice/experiment. Please click here to view a larger version of this figure.

Table 1: Number of successful UG-OTIM tumors compared to surgical implantation. Data combined from 2-4 independent experiments per high or low titer condition with n=5-10 mice per experiment. "Surgical injection" indicates mice that received abdominal laparoscopic surgical injection of 125,000 tumor cells. "Tumor-bearing" indicates mice with a pancreatic tumor. "Enrollable" indicates proportion of tumor-bearing mice at each time point with a tumor volume >20mm³. "Seeded Peritoneal Tumors" indicates the total proportion of tumor-bearing mice that had concomitant seeding of tumors on the peritoneal wall from tumor cell injection. Mice with tumors presenting incorrectly in tissues other than the pancreas (i.e., kidney) were excluded from "tumor-bearing" populations (but included in total injected mice). ND, not determined. Frequency of peritoneal wall seeding comparing high and low titer groups combined versus surgical implantation, p < 0.0029 via 2-tailed T test with Mann-Whitney post-test.

Figure 4: UG-OTIM tumors recapitulate the KPC tumor microenvironment. Representative images are shown. Top row, KPC tumors. Bottom row, UG-OTIM tumors at 5 weeks post-implantation. (A) Gross anatomy of excised tumors upon necropsy. (B) H&E (10X, bar=100µm) (C) Immunohistochemistry staining for CD3 (brown) (10X, bar=100µm). (D) Immunofluorescent staining of F4/80 (red) and DAPI (blue) (4X, bar=500µm). Please click here to view a larger version of this figure.