SURGERY ACCELERATES PANCREATIC LIVER METASTASIS IN AN IMMUNE COMPETENT MOUSE MODEL

Introduction:
Pancreatic Adenocarcinoma (PDAC) is highly lethal due to overwhelming metastatic burden. The mechanisms underpinning metastasis in PDAC are lacking. The TMEM doorway is the sole portal of intravasation in breast cancer (BC). Importantly, intravasation and dissemination via TMEM doorways is inhibited by Tie2 blockade in BC. We evaluated whether TMEM doorways are a common targetable mechanism of dissemination in PDAC.
Methods:
Tumor samples from treatment-naïve and neoadjuvant-treated patients who underwent curative-intent resection of PDAC were stained for TMEM doorways using a triple immunohistochemistry (IHC) stain (tumor cells expressing the actin regulatory protein Mena, macrophages expressing CD68, and endothelial cells expressing CD31). TMEM doorways were manually counted in the highest TMEM doorway-bearing 400x field (“hotspot”) identified at 100x scanning magnification for each case. TMEM doorway function was assessed in a murine model of PDAC treated with or without rebastinib (an investigational oral Tie2 inhibitor) by analyzing; [1] TMEM doorway associated vascular opening (TAVO) using high-molecular weight rhodamine dextran (155kD-TMR dextran), [2] circulating tumor cells (CTCs) and [3] disseminated tumor cells (DTCs) in murine livers. The Wilcoxon Test was used for the human TMEM doorway scoring comparisons. A student t-test was used for the murine analyses.
Results:
TMEM doorways were observed in all PDAC tumors from 50 unique treatment-naïve patients and 14 of 15 neoadjuvant-treated patients. The TMEM doorway score was ≈6x higher than observed in published BC patient samples. Mean TMEM doorway score in “hotspots” was 19 and 34 from low- and high- grade treatment-naïve tumors, respectively (p < 0.01). The mean TMEM doorway score was 3.1 in PDAC from neoadjuvant-treated patients. TAVO was decreased by rebastinib treatment (159.8 µm² ± sd26.5 vs. 22.6 µm² ± sd3.5, p < 0.01). PDAC tumor bearing mice treated with rebastinib had a 6.5-fold decrease in the number of CTCs compared to control (5554 ± sd1017 vs. 852 ± sd412 cells/mL blood, p < 0.01). PDAC tumor bearing mice treated with rebastinib had fewer DTCs in the liver compared to untreated mice (11.6 vs. 7.0 DTCs/mm²).
Conclusions:
TMEM doorways appear to be a common mechanism of dissemination in PDAC and BC. TMEM doorway concentration is significantly associated with PDAC tumor grade and decreases with neoadjuvant therapy. Inhibition of Tie2 with the selective inhibitor, rebastinib, preclinically leads to decreased TMEM doorway function and dissemination in PDAC. Tie2 is a promising therapeutic target for decreasing dissemination and potential metastasis in PDAC that warrants further clinical development.

Pancreatic ductal adenocarcinoma (PDAC) is a resistant malignancy with dismal outcomes. Early diagnosis, systemic treatment, and surgical resection are interdependently essential in improving survival. However, 20-30% of patients who undergo primary tumor resection will experience a metastatic recurrence in the liver within six months of surgery, despite no substantial differences in clinical history. This “rapid recurrence” (rrPDAC) is poorly understood. These metastatic lesions have a few possible origins, including occult synchronous metastases and disseminated metachronous lesions. We hypothesize metastases from either origin are accelerated by systemic and microenvironmental changes due to surgical intervention.
In RNA-seq of human rrPDAC primary tumors, we identified increased expression of Myc-targets when compared to long-term non-recurrers (no metastasis 18 months after surgery). Here, we describe a novel mouse model of immune competent, surgically resected PDAC that models rapid recurrence. Utilizing novel cell lines derived from our lab’s inducible, p48-Cre-recombinase driven LSL-KrasG12D/+ LSL-ROSA-MYC+/+ (KMC) mouse model, we orthotopically implanted KMC tumor cells into the pancreas of immune competent mice and monitored tumor growth and liver metastases weekly for two weeks via ultrasound. Mice taken down at day 14 (pre-surgery) did not demonstrate micrometastases on serial liver sectioning, however circulating tumor cells were present and isolated from venous blood. Experimental mice were randomized into control (n=24), distal pancreatectomy (n=17), and sham laparotomy cohort (n=11) and metastases were tracked via ultrasound twice-weekly. Surgically resected mice developed liver metastases 11 days earlier than controls (p <0.0001) and sham surgery mice developed liver metastases 10 days earlier than controls (p=0.02).
In the rrPDAC KMC model, RNA-sequencing of liver parenchyma of mice one day after surgery showed significantly elevated levels of Saa1/2 mRNA compared to nonoperative controls. Saa1/2 is indicative of a systemic inflammatory response after surgery, and Saa1/2 have previously been reported to contribute to the pre-metastatic niche development in pancreatic cancer (1), but this was reported in the context of normal PDAC development, not in a surgical setting. We hypothesize the systemic inflammatory response and corresponding increase in Saa1/2 after surgery primes the liver for metastatic outgrowth.
This model will allow for investigation into rrPDAC and the role surgery plays in exacerbation of metastasis in humans.

References:
Lee J et al. Serum Amyloid A Proteins and Their Impact on Metastasis and Immune Biology in Cancer. Cancers (Basel). 2021 Jun 25;13(13):3179.