Only 8% of patients diagnosed with pancreatic cancer today survive longer than 5 years despite aggressive interventions 1 . Although surgery can provide a clear therapeutic benefit for eligible patients, especially when combined with radiation and chemotherapy 2-4 , a sustained clinical response is rarely achieved in the majority of patients.
Our understanding of the underlying factors which contribute to disease recurrence following surgery is currently limited by the paucity of immunocompetent preclinical models of surgically resectable PDAC. The potential of such models to provide critical insights has been elegantly demonstrated in two recent publications which characterize the impact of (neo)adjuvant chemotherapy treatment on the immune system and tumor growth following surgical resection 5, 6 . However, widespread implementation of this model may be limited by the challenges associated with acquiring and maintaining the transgenic mouse strain and the in vivo electroporation procedures needed for initiating tumor growth.
In this report we describe the development of a surgically resectable orthotopic model of PDAC which reflects many of the hallmarks of human PDAC pathology which only requires the implantation of the well characterized, widely available Pan02 cell line. Additionally, we demonstrate that this model can be used to investigate the effects of surgery on a clinically relevant immunotherapy.
Pan02 murine pancreatic adenocarcinoma cell line 7 
John Bell (OHRI, Ottawa), and was maintained in DMEM (Corning Cellgro 10-013-CV) supplemented with 10% FBS (Seradigm #1500-500). B78H1GM cells were obtained from Dr Elizabeth Jaffee (Johns Hopkins, Baltimore). Cells used during this study were less than passage 30 and confirmed to be free of mycoplasma contamination by Hoescht staining (Thermofisher Cat#62249) as per manufacturer's protocol.
Female C57BL/6 mice 6 weeks of age were purchased from Charles River Laboratories. Male animals were not included in the current study as sex is not anticipated to impact the surgical procedure or outcomes. Animals were housed in pathogen-free conditions and all studies performed were in accordance with institutional guidelines at the Animal Care Veterinary Service facility of the University of Ottawa (Ontario). The Canadian Council on Animal Care and the Animal Care Committee of the University of Ottawa approved this study. Mice were euthanized by intraperitoneal (IP) injection of Pentobarbital Sodium (65mg/kg).
Routine perioperative care (including anesthesia and pain management) was performed as previously described 8 . Briefly, mice were subjected to 2.5% isofluorane (Baxter Corp.) for induction and maintenance of anesthesia. Hair was removed from the surgical site with Nair TM or by shaving. For all surgical procedures, the surgical site and surrounding areas are prepared with Betadine®️ surgical scrub, 70% alcohol, and Betadine®️ surgical paint. Following a mid-line incision (1-3 cm) the tail of the pancreas was identified and isolated using a cotton swab moistened with saline. A 30G ½ inch needle (Hamilton, Reno 7655-07, Model 1705RN syringe, Hamilton 7803-07, length 0.625" needle) was used to inject Pan02 cells in a total volume of 10 μ l of PBS into the tail of the pancreas. Following removal of the needle the injection site was inspected to ensure no leakage prior to cleaning the injection site with saline and returning the pancreas to the abdominal cavity. The underlying muscle and skin are then sutured using 5.0
Polysorb™ absorbable suture (Covidien) or 9mm stainless steel wound clips (BrainTree Scientific) respectively. The clips were removed 7 days following tumor injection.
(Supplementary Video 1)
Primary tumors were resected by laparotomy on day 15 (1x10 5 cells implanted) or day 20 (1x10 3 and 1x10 4 implanted cells). Briefly, mice are anesthetized and prepared as described above. A 1-1.5 cm midline incision is then made starting at the umbilicus and cutting upward towards the sternum through the skin and muscle along the linea alba to ensure minimal bleeding. The tail of the pancreas and adjacent area is then located and isolated with sterile saline soaked cotton swab.
The tumor along the splenic portion of the pancreas is isolated from the splenic artery using sutures and removed, keeping the mesenteric pancreas intact using sutures (5.0 Polysorb™ absorbable sutures (Covidien) or PDS II suture (Ethicon Z423)). Any bleeding observed in the abdomen was located and sutured (arteries) or cauterized to limit mortality post-surgery. The surgical incision was closed as described above using Polysorb™ sutures and staples. counterstained with hematoxylin. All samples were analyzed by a certified pathologist for tissue identification, analysis and validation.
Pancreatic tumors were collected following surgical resection, and were placed in dissociation media consisting of RPMI supplemented with 10% FBS, Collagenase I and DNase IV. 
The entire contents of the peritoneal cavity including tumor tissue was removed and weighed and normalized to body weight (peritoneal weight / total body mass x 100%) to determine the overall tumor burden present.
The viability and functionality of splenocytes isolated 20 days following partial pancreatectomy were assessed in mice receiving an initial injection of 1x10 4 Pan02 cells. Briefly, viability of splenocytes isolated as previously described 10 
One-way analysis of variance (ANOVA) with Tukey post hoc tests or Student's T test were performed for all data. A P-value of <0.05 was considered significant. Survival was plotted as a Kaplan-Meier survival curve and statistical differences assessed by log-rank tests.
tumor with development of metastatic peritoneal disease.
To develop a surgically resectable murine model of PDAC which recapitulates human disease we first sought to determine the optimal number of Pan02 that could be orthotopically implanted into the pancreas and yield a resectable primary tumor. Total cell numbers of 1x10 3 , 1x10 4 or 1x10 5 were injected into the tail of the surgically exposed pancreas and the tumor burden examined at 15 and 20 days post seeding ( Figure 1 and Table 1 ). Notably, tumour outgrowth was present at the injection site in the majority of mice at all injection doses ( Figure 1B -D).
Peritoneal disease was also visibly absent in the majority of mice receiving 1x10 3 established histological features associated with invasive carcinoma as described by Hruban and Fukushima 11 in 9 of 10 animals suggesting that orthotopic injection of Pan02 cells into the tail of the pancreas results in a pancreatic disease representative of human pathology ( Figure 2 ).
Lymphocytic infiltration is strongly correlated with patient outcomes in PDAC 12-14 therefore we also sought to characterize the immune cell populations present within the orthotopic tumor ( Figure 3 ). Interestingly, CD3 + cells were consistently observed within the cancerous regions of the pancreas but were rarely found within the healthy pancreatic tissue ( Figure 3A , compare tumor (T) with healthy pancreatic tissue (P)). Subsequent staining revealed that both CD4 + and CD8 + cells contribute to the CD3 + population found within the TME. Flow cytometric analysis of dissociated tumors further revealed that CD4 + and CD8 + T cells were present in nearly equal proportions suggesting the presence of an active immune response to the growing tumor ( Figure   3B ). Interestingly, a significant proportion of the infiltrating CD4+ population also expressed markers of regulatory T cells suggesting the presence of an immunosuppressive tumor microenvironment (TME) (Tregs, CD4 + Foxp3 + CD25 + , p=0.0004 over control mice, Figure 3C ).
Consistent with this observation CD8 + T cells found within the TME consistently exhibited increased expression of PD-1 compared to circulating T cells (p=0.01) ( Figure 3D ). Further examination of the immune infiltrate also revealed the presence of additional cell populations with potentially suppressive activity including myeloid derived suppressor cells (CD11b + Gr1 + ) and a large number of CD19 + B cells which could possibly represent regulatory B cells 15 ( Figure   3B ).
Having characterized the conditions necessary for establishing resectable orthotopic tumors we next aimed to determine whether this model could be used to identify parameters contributing to 1 1 the success of surgical intervention. Surgical resection of the primary tumor on day 15 or 20 post injection for mice receiving either 1x10 5 or 1x10 4 Pan02 cells respectively ensured the resection could be performed in the absence of visible peritoneal disease, splenic metastases, or extensive adherence to organs as is generally required in the clinical setting 16 ( Figure 4A and Table 1 ).
However, despite surgical resection of the primary tumor, disease was evident in both the pancreatic remnant and disseminated throughout the peritoneal cavity by day 40 ( Figure 4B and Table 1 ). The total disease burden, as quantified by normalizing peritoneal weight to total body mass, was significantly increased compared to a control cohort ( Figure 4C Table 2) .
In addition, we also sought to examine whether splenic immune function, in particular viability and T cell functionality, could be maintained following surgery in this model since clinical evidence supports a beneficial role of spleen preservation in improved outcomes and decreased complications [18] [19] [20] [21] . Histological examination of the spleen indicates that surgical resection of the primary tumor does not noticeably impact splenic architecture as the white and red pulp, marginal zones and follicles were still present ( Figure 5A , n=10 total number of mice examined).
However, an increased splenic weight and an accumulation of erythrocytes within the red pulp were observed in mice following resection ( Figure 5A and B). The apparent accumulation of blood is consistent with a reduced drainage through the splenic vein which was severed as part of our tumor resection procedure. However, despite evidence of blood accumulation, there was no significant difference in splenocyte viability between surgery and non-surgery groups ( Figure   5C , n=3 vs n=5, p=0.744). Finally, no significant difference in TNFα secretion from PMA/ionomycin stimulated T cells was evident ( Figure 5D , n=3 v n=5, p=0.4) suggesting the function of CD8 + T cells was unaltered following surgical resection.
Next, we sought to determine whether surgical resection could improve survival and disease recurrence in the orthotopic Pan02 model. At the time of surgery, none of the animals had visible signs of peritoneal disease and gross tumour resection was complete. Although an initial reduction in body weight occurred in the immediate perioperative period the animals undergoing surgery gained weight at a similar rate during the subsequent weeks ( Figure 6A ). Surgery was not found to provide any survival benefit when compared to the non-surgery group with disease evident at both the site of the primary tumor and disseminated throughout the peritoneal cavity with metastases observed in the spleen and liver ( Figure 6 and Table 3 ).
Accumulating clinical data indicates that neoadjuvant therapies can improve PDAC surgery patient outcomes 22 25 . GVAX in combination with Treg depletion has previously been shown to provide a therapeutic benefit in preclinical models through immune mediated mechanisms 9, 26 . To determine whether the immunosuppressive effects of surgical stress significantly impact the efficacy of GVAX we resected orthotopic tumors according to the schedule outlined ( Figure 7A ).
In agreement with previous findings, GVAX treatment alone significantly improved survival without tumor resection ( Figure 7B) . Furthermore, the therapeutic benefit of GVAX was not significantly impacted by surgery.
Surgical resection is currently the most effective treatment strategy for patients with an operable pancreatic tumor 27 The similar outcomes between these groups suggests immunotherapies like GVAX may not provide additional benefits in the surgical setting despite the significant tumor debulking associated with surgical resection. It is unclear whether this is a consequence of the 1 5 immunosuppressive environment associated with surgical stress 10, 30, 31 or other factors associated with PDAC. However, this result provides a proof-of-concept and further emphasizes the need for models that accurately depict surgical resection of PDAC so that the timing, dose and full efficacy of combination therapies can be adequately explored prior to initiating clinical trials.
In summary, this orthotopic model recapitulates many aspects of the human disease and requires no specialized equipment or surgical procedures allowing easy adoption by other laboratories interested in assessing the effects of perioperative therapies in PDAC. 
Animal studies complied with the Canadian Council on Animal Care guidelines and were approved by the University of Ottawa Animal Research Ethics Board (ME-1664).
Not applicable
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
The authors declare that they have no competing interests.
Innovation Award (703424). The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
All 