Establishment of Patient-derived Orthotopic Xenografts (PDX) as Models for Pancreatic Ductal Adenocarcinoma

Abstract

Background/Aim: Pancreatic cancer (PC) is one of the leading causes of cancer-related death. The purpose of the present study was to establish a patient-derived orthotopic xenograft model (PDOX) for pancreatic ductal adenocarcinoma (PDAC), thus providing a tumor microenvironment resembling that of the human pancreas to identify novel potential biomarkers and treatment regimens.

Materials and Methods: PDAC tissue samples were received from 35 patients, following informed consent, and three mouse strains were implemented.

Results: Successful PDOX engraftment was performed in nonobese diabetic/severe combined immunodeficient (NOD/SCID) and NOD/SCID gamma (NSG) mice. Nonetheless, we found a higher rate of successful engraftment and tumor growth in NSG compared to NOD/SCID mice, possibly owning to the different level of immunosuppression and more specifically of the natural killer cells presence.

Conclusion: Our suggested PDOX model represents a preclinical cancer research model with a high affinity for the patient’s tumor microenvironment, thus enabling the acceleration of PDAC research.

Pancreatic cancer (PC) is one of the leading causes of cancer-related death and the fourth cause of cancer mortality in the USA (1,2). Most of the cases diagnosed with PC are ductal adenocarcinomas (PDACs), most frequently located in the head of the pancreas (3,4) and associated with poor prognosis (5). Depending on the degree of differentiation and the tumor microenvironment, the malignancy may present poorly to well-formed glands or infiltrating cells forming sheets (3,4). Besides the great research efforts, the mortality rate regarding PC is increasing steadily, thus being projected that by 2030 it will represent the second cancer-related cause of mortality (6).

In the same context, it is well accepted that tumor development and growth depend on the tumor microenvironment and metabolism (7). Patient-derived xenograft (PDX) models represent the missing link, enabling the examination of tumor tissue in a native environment without significantly affecting the heterogeneity, genomics, and stromal architecture of these neoplasms (8). Originally, the subcutaneous approach was established as the gold-standard method for PDX implantation and growth, but with certain limitations (9). In fact, the subcutaneous injection of PDX samples into mouse flanks may fail to generate the appropriate tumor microenvironment, thus affecting tumor growth and clonal heterogeneity (9). To face this challenge, we established a patient-derived orthotopic xenograft (PDOX) model for PDAC as a preclinical model, providing a tumor microenvironment resembling that of the actual patient pancreatic cancer.

Materials and Methods

Animals. Male nonobese diabetic/severe combined immunodeficient (NOD/SCID) (NOD.Cg-Prkdc^scid/J, JAX stock #001303), NOD/SCIDgamma (NSG, NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ, JAX stock #005557), and RAG-1 deficient (RAG, B6.129S7-Rag1^tm1Mom/J, JAX stock #002216) mice. These three mouse strains represent different levels of immune deficiency with the NSG mice considered as the mice with the highest immunodeficiency. Briefly, NOD/SCID mice are homozygous for the severe combined immune deficiency spontaneous mutation (Prkdc^scid) and are characterized by an absence of functional T cells and B cells and some NK cell functions (normal antigen-presenting cell, myeloid, and NK functions as though strain-dependent) (10). NSG mice were produced by breeding female NOD.CB17-Prkdc^scid/J mice with male mice bearing the X-linked B6.129S4-Il2rg^tm1Wjl/J allele and thus carry two mutations on the NOD/ShiLtJ genetic background: severe combined immune deficiency (scid) and a complete null allele of the IL2 receptor common gamma chain (IL2rg^null). The scid mutation is in the DNA repair complex protein Prkdc and renders the mice B and T cell deficient. The IL2rg^null mutation prevents cytokine signaling through multiple receptors, leading to a deficiency in functional NK cells (11). Finally, RAG-1 mice are homozygous for the Rag^1tm1Mom mutation and produce no mature T cells or B cells due to the inability to perform V(D)J recombination (12).

All mice were of 6-8 weeks age, were housed in a specific pathogen-free (SPF) environment under controlled conditions regarding light (12 h cycle), humidity (~50-60%), and temperature (20-22˚C), and allowed food and water ad libitum.

The handling and experimentation of the animals were conducted in accordance with the Greek laws (PD 56/2013 and Circular 2215/117550/2013) and the guidelines of the European Union (2013/63/EU) under a licensed protocol approved by the IACUC and Greek authorities (License no. 5542/228006).

Patient consent and ISB approval. The study prospectively enrolled 35 consecutive patients with PDAC who underwent surgery at the Department of Surgery, University Hospital of Larissa, Greece (from 5/2017 to 5/2020). Patients’ characteristics are demonstrated in Table I. All patients provided written informed consent. Upon the excision of the pancreatic tumor, either by a Whipple procedure (tumor located at head/neck) or by a distal pancreatectomy and splenectomy (tumor located at tail), tumor samples were obtained by experienced pathologists. In cases of nonresectable tumors with distant metastases, biopsies were received from the most easily accessible metastatic lesions. The study protocol was approved by the Institutional Scientific Board of the UHL (Approval number of ISB: 20053/26-4-17).

Table I. Patient characteristics.

n: Number; ERCP: endoscopic retrograde cholangiopancreatography.

Establishment of patient-derived orthotopic cancer xenografts. The surgically excised tumor tissue samples were transferred into a 60 mm petri dishes, washed in PBS 1X and finely minced using sterile scalpels to obtain ~1-3 mm³ pieces. Tumor fragments were further processed via enzymatic dissociation in tissue dissociation solution (TDS) containing a mix of Hyaluronidase, Collagenase IV, and DNase I in HBSS supplemented with 10% FBS. Samples were incubated in TDS at 37˚C for 20-30 min (1 ml/100 g tissue). To ensure that an equivalent number of cells was injected into each mouse and minimize variability in tumor size, tumor samples were dissociated into single cell suspensions by either filtered through a 100 μm cell strainer or forced to pass through a 16G needle syringe. The cell suspension was then pelleted (1,400 rpm, 10 min), washed in PBS 1X, resuspended in serum-free RPMI, mixed with Matrigel^© Matrix, and injected orthotopically into mice.

Orthotopic tumor implantation. For the development of the orthotopic xenografts, mice were anesthetized by inhaling 2% isoflurane in 100% oxygen. Before proceeding with the operation, mice were tested to ensure they were fully anesthetized. As soon as full anesthesia occurred, a small left lateral incision (1 cm) was performed and following the opening of the peritoneum, the pancreas was exposed and retracted (Figure 1). Ingestion of 10 μl of the Matrigel solution including the minced tissue was performed axially to the body of the pancreas with direction from the tail to the head (Figure 1). Finally, the abdomen was closed in two layers (peritoneum, skin). The peritoneum was closed using 4-0 polyglycolic acid rapid suture in a continuous manner and the skin was closed using staples. Finally, 10 μl of meloxicam was administered subcutaneously for analgesia. Mice were left to recover over heated pads before being transferred back to the animal facility. As soon as mice recovered, they were transferred back to the animal facility where they were monitored daily for signs of pain and distress. To avoid post-surgery pain, mice were administered subcutaneously with meloxicam. Then, mice were observed daily for the appearance of signs of tumor development in the area of the operation. When tumors were apparent by palpation, mice were euthanized to further examine pancreata, ensure the development of tumors, and weigh the excised tumors.

Figure 1. Basic steps for the patient-derived orthotopic xenograft (PDOX) engraftment. (A) Skin and peritoneum incision, (B) orthotopic ingestion of PDOX solution in the pancreas, (C) closure of the peritoneum and skin.

Statistical analysis. A Chi-square test was performed to compare categorical variables between the two periods. Comparisons of continuous variables were performed with a two-tailed unpaired t-test for parametric data and Mann-Whitney U-test for nonparametric data. Kruskal-Wallis test was used for non-parametric comparison of outcomes regarding NOD/SCID, NSG, and RAG mice. Differences were considered significant with a p≤0.05. Descriptive statistics were performed using Prism Graphpad 9.3 (GraphPad Software, San Diego, CA, USA).

Results and Discussion

A total of 40 implantations of PDAC PDOX were performed in NSG, 10 in NOD/SCID, and 6 in RAG mice (Table II). In Figure 2 we demonstrate the difference in pancreatic tissues between a PDOX model and a normal control animal. No difference was reported regarding the postoperative survival rate (Table II, Figure 3). Nonetheless, there was a significant difference regarding the successful engraftment rate (Table II, Figure 3). In fact, the engraftment rate was higher in NSG mice (97.3%) compared to NOD/SCID (57.1%) and RAG (0%) mice (p<0.01). In the same context, NSG presented also the shortest period from engraftment to tumor excision (p<0.01) (Table II, Figure 3). In addition, mean tumor weight and size were lower in the NOD/SCID group compared to the NSG mice (Table II, Figure 3). No distant metastases were found.

Table II. Endpoints of PDX engraftment in three different mouse strains.

n: Number; PDX: patient-derived xenograft; SD: standard deviation; NSG: NOD/SCID gamma; NOD/SCID: nonobese diabetic/severe combined immunodeficient; RAG: RAG-1 deficient.

Figure 2. Differences regarding pancreatic tissues between a patient-derived orthotopic xenograft model and normal control. (A) Engrafted tumor in the anatomic area of the pancreas, (B) size of the excised tumor and pancreatic tissue, (C) weight of tumor and the pancreas, (D) normal pancreas, (E) size of normal pancreas, (F) weight of normal pancreas.

Figure 3. Graphs demonstrating differences in (A) engraftment rate, (B) median days to excision, (C) median tumor weight, and (D) median tumor size of patient-derived orthotopic xenograft (PDOX). A) NSG strain demonstrated a significantly higher engraftment rate. B) The violin plot shows a significantly shorter time-period to excision of the tumor for the NOD/SCID gamma (NSG) strain. C) The violin plot demonstrates a higher median tumor weight for the NSG strain. D) The violin plot demonstrates a higher median tumor weight for the NSG strain. *p-value<0.05, **p-value<0.01; ***p-value<0.001.

The successful development of PDOX has already been described at the beginning of the 90’s in the work published by Furukawa et al. that reported the development of clinically relevant orthotopic models from human gastric cancer by implanting histologically intact tumor tissues in nude mice (13). Since then different PDOX models have been implemented as preclinical models of cancer in various types of cancer (14-17). The results of the current report showed that pancreatic cancer PDOX models are feasible and efficient. PDOX models are reported and expected to better recapitulate human tumors compared to the subcutaneous PDX models (18), while they better evaluate targeted anti-metastatic therapies (19). Nonetheless, the outcomes of the current study demonstrated that the rate of successful engraftment and tumor growth is affected by the level of immunodeficiency. Even though previous studies have found the impact of different surgical and oncological factors on the successful engraftment of PDOX (20), herein we also demonstrated the significance of the level of immunosuppression on the engraftment success. NSG mice that are characterized by a lack of T, B, and natural killer (NK) cells (triple deficient) (21), were the most immunodeficient of the three strains that were used in the experiments. Interestingly, we found that PDOX in NSG mice was associated with the highest engraftment rate and tumor growth. NOD/SCID are considered to be less immunodeficient compared to NSG mice, because of the presence of partially functional NK cells (22), and this could be the reason for the lower engraftment rate, along with the lower tumor growth. Finally, RAG-1 mice, which presented the least immunodeficient status, only lacking mature B and T lymphocytes (23), did not grow any pancreatic tumors. These findings highlight the significant role of immune surveillance against cancer cells, which represents a certain hallmark of cancer (24). In fact, our study suggests the significant impact of NK cells deficiency in tumor growth and metastasis at least in the case of PDAC. Previous studies have implicated the dysfunction and under-regulation of NK cells, thus suggesting the potential value of intervening at the stage of no or minimal disease (25). On this basis, certain NK-cell-based treatment strategies have been proposed for PDAC, including receptor-mediated activation and ex vivo expansion of NK cells, along with the chimeric antigen receptor engineering (CAR-NK) (26). Furthermore, NK cell checkpoint inhibitors have been developed, such as Lirilumab (IPH2102/BMS-986015), but its value in terms of survival endpoints has yet to be demonstrated in human trials (27).

Unfortunately, we did not observe any distant metastasis in any of the PDOX developed in our lab, which is in agreement with a recently published work by Pham et al. (28). However, we must underline a potential limitation of this study as we did not use fragments of tumor tissue but rather cell pellets to develop the PDAC-PDOX. The use of tumor fragments instead of cell pellets has been reported previously to result in a superior quality of PDOX, which mimic better the patient, as they show a higher potential to metastasize (18,19). It is noteworthy that such an approach can keep, at least at the initial steps of the development of the PDOX, the exact architecture of the tumor, retaining also the tumor microenvironment intact and resulting in a more clinical relevant PDOX as has already been demonstrated by the group of Hoffman (29). Taking into account the significant role of desmoplasia and cancer-associated fibroblasts (CAFs) especially in PDAC (30,31) it would be of great importance to keep the architecture of the tumor as intact as possible during the development of realistic and more useful PDX. In the context of these issues that have been raised for the development of more clinically relevant PDOX, we suggest that a comparison of different implantation approaches along with the different immunodeficient mouse strains available nowadays (e.g., cell pellet vs. fragments of histologically intact tumor tissues vs. tumor organoids) would be of great interest.

To summarize, as the research for new treatment regimens and immunotherapies for PDAC progresses, the orthotopic PDX model we present herein enables the in-depth evaluation of new drugs in a tumor microenvironment much closer to that of the actual tumor, thus accelerating cancer research and allowing the development of more potent therapeutic approaches. Further studies to improve these models to better mimic the actual clinical situation are ongoing in our department.

Conflicts of Interest

Dr. Dimitrios Magouliotis, Kostas Lafazanis, Fani Koutsougianni, Nikos Sakellaridis, Maria Ioannou, Dimitris Zacharoulis, Konstantinos Dimas declare no conflicts of interest regarding this study.

Authors’ Contributions

DEM contributed to the conception and design of the work, the experiments, the acquisition, analysis, and interpretation of data for the work, the drafting of the work and revising it critically for important intellectual content, the final approval of the version to be published, and is accountable for all aspects of the work.

KL and FK contributed to the experiments, the interpretation of data for the work, the revising of the work critically for important intellectual content, the final approval of the version to be published, and is accountable for all aspects of the work.

MI contributed to pathologic examination of the tissue, the acquisition and analysis of data for the work, the drafting the work and revising it critically for important intellectual content, the final approval of the version to be published, and is accountable for all aspects of the work.

NS, DZ, and KD contributed to the design of the work, the supervision of the experiments, the acquisition and analysis of data for the work, the drafting the work and revising it critically for important intellectual content, the final approval of the version to be published, and is accountable for all aspects of the work.

Acknowledgements

This research has been co-financed by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH–CREATE–INNOVATE (project code: T1EDK-01612).