Impaired Synthesis of Stromal Components in Response to Minnelide Improves Vascular Function, Drug Delivery, and Survival in Pancreatic Cancer

Abstract

Purpose: Pancreatic cancer stromal microenvironment is considered to be the major reason for failure of conventional and targeted therapy for this disease. The desmoplastic stroma, comprising mainly collagen and glycosaminoglycans like hyaluronan (HA), is responsible for compression of vasculature in the tumor resulting in impaired drug delivery and poor prognosis. Minnelide, a water-soluble prodrug of triptolide currently in phase I clinical trial, has been very effective in multiple animal models of pancreatic cancer. However, whether Minnelide will have efficacious delivery into the tumor despite the desmoplastic stroma has not been evaluated before.

Experiment design: Patient tumor-derived xenografts (PDX) and spontaneous pancreatic cancer mice were treated with 0.42 and 0.21 mg/kg body weight for 30 days. Stromal components were determined by IHC and ELISA-based assays. Vascular functionality and drug delivery to the tumor were assessed following treatment with Minnelide.

Result: Our current study shows that treatment with Minnelide resulted in reduction of ECM components like HA and collagen in the pancreatic cancer stroma of both the spontaneous KPC mice as well as in patient tumor xenografts. Furthermore, treatment with Minnelide improved functional vasculature in the tumors resulting in four times more functional vessels in the treated animals compared with untreated animals. Consistent with this observation, Minnelide also resulted in increased drug delivery into the tumor compared with untreated animals. Along with this, Minnelide also decreased viability of the stromal cells along with the tumor cells in pancreatic adenocarcinoma.

Conclusions: In conclusion, these results are extremely promising as they indicate that Minnelide, along with having anticancer effects is also able to deplete stroma in pancreatic tumors, which makes it an effective therapy for pancreatic cancer.

Figure 1

Figure showing presence of extensive stroma in pancreatic ductal adenocarcinoma in (A) patient tumor derived xenografts (PDX) and (B) spontaneous KRasG12D,TP53,Pdx-Cre (KPC) mice in H&E stain, picrosirius red stain. Normal pancreas tissue showed very little staining with picrosirius red (C).

Figure 2

Minnelide reduces stromal component alpha SMA and HA in (A) patient tumor derived xenograft as well as (B) in KPC tumors. (C) Picosirius red staining is also decreased in both models. (D) Quantitation of HA from the PDX tumors (E) and KPC tumor (F) also shows decreased HA in the tumors. The * represents p<0.05.

Figure 3

Minnelide decreased hydroxylysine in (A) patient tumor derived xenografts and (B) KPC tumors; hydroxyproline in patient tumor derived xenografts (C) and (D) KPC tumors; and alpha Ketoglutarate in (E) patient tumor derived xenografts and (F) KPC tumors. The * represents p<0.05.

Figure 4

Minnelide decreased (A) HA synthase expression along with (B) the activity in KPC tumors. (C) Hyaluronidase expression as well as (D) activity was found to be unaltered in these tumors following treatment. The * represents p<0.05.

Figure 5

Minnelide improved (A) drug delivery as seen by doxorubicin fluorescence in both KPC and PDX tumors. Minnelide also reduced vascular compression (B) and resulted in (C) more “open” blood vessels compared to untreated tumors. This resulted in a (D) greater survival of the KPC mice receiving Minnelide compared to the untreated mice. The * represents p<0.05.

Figure 6

Triptolide decreased viability of cancer-associated fibroblasts as well as epithelial tumor cells. Representative CAFs from KPC tumors, K-CAF1 (A), K-CAF5 (B) and epithelial mouse PDAC cell lines KPC001 (C) and Panc02 (D) were treated with indicated doses of triptolide for 24 and 48h. Viability was plotted as percent of untreated control. The IC50 values are represented in Supplementary table 2.