Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Summary

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

Abstract

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.

Introduction

PCa, and its most common form, the Pancreatic Ductal Adeno Carcinoma (PDAC), is one of the most common causes of cancer-related death with a 1-year survival rate lower than 20% and a 5-year survival rate of 8%, regardless of the stage^1,2. The disease is almost always fatal, and its incidence is forecasted to continuously grow in the next years, unlike other cancer types, whose incidence is declining³. Factors such as late cancer detection, the tendency of rapid progression, and lack of specific therapies lead to a poor prognosis of PCa⁴. Great advances in cancer research have been obtained, thanks to the development of more accurate preclinical mouse models. The models have provided appropriate insights to the understanding of the molecular mechanism underlying cancer and to the development of new treatments⁵. These advances poorly apply to PCa, which, despite great recent efforts, remains resistant to current chemotherapeutic therapies¹. For these reasons, the development of novel approaches to improve patients' prospects is mandatory.

Over the years, many PCa mouse models have been developed, including xenografts, which are the most widely used models nowadays⁵. Xenograft models are classified as subcutaneous heterotopic and orthotopic, depending on the location of the implanted tumor cells. Subcutaneous heterotopic xenografts are easier and cheaper to accomplish but miss certain characteristic features of PCa (i.e., the peculiar tumor microenvironment, characterized by the accumulation of fibrotic tissue, hypoxia, acidity, and angiogenesis)^6,7. This explains why subcutaneous xenografts often fail to provide robust data for therapeutic treatments leading to failures when translated to the clinical setting⁸. On the other hand, orthotopic xenografts resemble the tumor microenvironment more closely, leading to better mimicking of the natural development of the disease. In addition, orthotopic xenografts are more suitable for studying the metastatic process and the invasive features of PCa, which almost do not occur in subcutaneous models⁹. Overall, orthotopic xenograft mouse models are nowadays preferred to perform preclinical drug testing^9,10. Orthotopic xenografts usually rely on surgical procedures to implant either cells or very small tumor tissue pieces into the pancreas. Indeed, several papers based on surgical models of PCa have been published in the last few decades¹¹. However, the quality and the outcome of the surgical procedure for the establishment of an orthotopic tumor model strongly depend on the technical skill of the operator. Another key point for a successful orthotopic PCa xenograft for a translational clinical approach is the possibility to establish localized disease with predictable growth kinetics.

To address these issues, here we describe an innovative procedure to produce an orthotopic PCa xenograft, exploiting ultrasound (US)-guided injection of human PCa cells into the tail of the pancreas in immunodeficient mice. This procedure generates a reliable PCa mouse model. The tumor growth is followed in vivo by US imaging.

Protocol

The present protocol received approval from the Italian Ministry of Health with the authorization number 843/2020-PR. In order to ensure aseptic conditions, the animals were maintained inside the barrier room of the research animal vivarium (Ce.S.A.L.) of the University of Florence. All procedures were performed in the same space where the mice were housed at the LIGeMA facility of the University of Florence (Italy). 1. Cell preparation Culture PCa cells from the P…

Representative Results

Following the protocol described above, mice were first anesthetized in an isoflurane chamber, and placed on the animal platform (Figure 1A). The pancreas was visualized with ultrasound imaging (Figure 1B). A 50 µL Hamilton syringe was loaded with 1 x 106 PANC1 cells suspended in 20 µL of PBS and placed on the needle holder (Figure 1C). The optimal angle between the syringe and the US transducer was 45° (<s…

Discussion

Although the use of US imaging is widespread in the clinic, tumor development in many preclinical mouse models is usually described using bioluminescent imaging¹¹. The latter is an indirect way to evaluate tumor engraftment and expansion and it also does not provide a reliable tumor growth kinetics. In the present study, we have applied US imaging for performing cell injection as well as for monitoring tumor development. The protocol we have described and the results we have provided represent a r…

Materials

100 mm Petri dish	Sarstedt, Germany	P5856
3D-Mode package	Visualsonics Fujifilm, Italy		Includes the 3D Motor; necessary for volumetric imaging
Aquasonic 100, Sonypack 5 lt Ultrasound Transmission Gel	PARKER LABORATORIES, INC.	150	Gel for ultrasound
Athymic Mice (Nude-Foxn1nu)	ENVIGO, Italy	69	20 females, 8 weeks old, Athymic Nude-Foxn1nu, 20-22 g body weight
CO2 Incubator Function Line	Heraeus Instruments, Germany	BB16-ICN2
Display of ECG, Respiration Waveform and body temperature	Visualsonics Fujifilm, Italy	11426
DMEM (Dulbecco’s Modified Eagle Medium)	Euroclone Spa, Italy	ECM0101L
DPBS (Dulbecco’s Phosphate Buffered Saline)	Euroclone Spa, Italy	ECB4004L
Eppendorf (1.5mL)	Sarstedt, Germany	72.690.001
FBS (Fetal Bovine Serum)	Euroclone Spa, Italy	ECS0170L
Hamilton Needle Pointstyle 4, lenght 30 mm, 28 Gauge	Permax S.r.l., Italy	7803-02
Hamilton Syringe 705RM 50 µL	Permax S.r.l., Italy	7637-01
Isoflo (250 mL)	Ecuphar	7081219
L-glutamine 100X	Euroclone Spa, Italy	ECB3000D
Mouse Handling table II	Visualsonics Fujifilm, Italy	50249
MX550D: 55 MHz MX Series Transducer	Visualsonics Fujifilm, Italy	51069	Ultrasound Transducers
Oxygen/isofluorane mixer	Angelo Franceschini S.r.l.	LFY-I-5A
PANC1 cell line	American Type Culture Collection (ATCC), USA	CRL-1469
Rimadyl (carprofen)	Pfizer	11319	20 mL, injection solution
Trypsin-EDTA 1X in PBS	Euroclone Spa, Italy	ECB3052D
Vet ointment for eyes, Systane nighttime	Alcon	509/28555-1
Vevo Compact Dual Anesthesia System (Tabletop Version)	Visualsonics Fujifilm, Italy	VS-12055	complete with gas chamber
Vevo Imaging Station 2	Visualsonics Fujifilm, Italy	VS-11983	Imaging WorkStation 1 plus Imaging Station Extension with injection mount
Vevo Lab	Visualsonics Fujifilm, Italy	VS-20034	Data Analysis Software
Vevo LAZR-X Photoacoustic Imaging System	Visualsonics Fujifilm, Italy	VS-20054	Includes analytic software package for B-mode
Vevo Photoacoustic Enclosure	Visualsonics Fujifilm, Italy	53157