Ex Vivo Testing of Patient-Derived Xenografts Mirrors the Clinical Outcome of Patients with Pancreatic Ductal Adenocarcinoma

Abstract

Purpose: Translation of the patient-derived xenograft (PDX) model into a method for practical personalized cancer treatment is prevented by the intense resources and time necessary to generate and test each tumorgraft. We aimed to develop a high-throughput ex vivo drug testing approach that can be used for personalized cancer treatment design.

Experimental design: We developed a unique ex vivo live tissue sensitivity assay (LTSA), in which precision-cut and uniform small tissue slices derived from pancreatic ductal adenocarcinoma PDX tumors were arrayed in a 96-well plate and screened against clinically relevant regimens within 3 to 5 days. The correlation between the sensitivities of tissue slices to the regimens and patients' clinical responses and outcome were statistically analyzed. The results of LTSA assay were further confirmed with biochemical methods in vitro and animal PDX model in vivo RESULTS: The ex vivo tissue slices remain viable for at least 5 days, and the tumor parenchyma, including stroma, vascular structures, and signaling pathways, are all retained. The sensitivities of the ex vivo tissue slices to gemcitabine and irinotecan was consistent with the clinical responses and outcomes of the patients from whom the tumorgrafts were derived (r = 0.77; P = 0.0002). Retrospective analysis showed that the patients who received LTSA-sensitive regimens had remarkably longer progression-free survival than patients who received LTSA-resistant regimens (16.33 vs. 3.8 months; n = 18, P = 0.011).

Conclusions: The results from these PDX and LTSA methods reflect clinical patients' responses and could be used as a personalized strategy for improving systemic therapy effectiveness in patients with pancreatic cancer. Clin Cancer Res; 22(24); 6021-30. ©2016 AACR.

Figure 1

Development of a high throughput ex vivo live tissue slice sensitivity assay (LTSA). (A) Technique flow of ex vivo tissue slice culture and drug sensitivity testing. PDX tumors were grown in nude or NSG mice; tissue cores were taken from harvested tumors and embedded in agarose gel; tissue slices were cut with a microtome machine and arrayed in 96 well plates; after 48-72 hours treatment, 10 μl Prestoblue agent were added into each well; after incubation for 2 hours, fluorescence intensity was read with plate reader. Inhibition of tissue slice viability by the agent, for example, auranofin (AUR) was analyzed through normalized the value of treated tissue slice with untreated control. (B) Ex vivo tissue slice cultures remain viable. Tissue slices generated from xenograft tumors were cultured in 96-well plates with 200 μl RPMI1640 medium, and tissue slice viabilities were measured with PrestoBlue every day as indicated. (C) Ex vivo tissue slice cultures retain microenvironmental structures of xenograft tumors. Tissue slices generated from xenograft tumors were cultured in 96-well plates with 200 μl RPMI1640 medium, and tissue slice viabilities were measured with PrestoBlue every day as indicated. The viability at Day-0 was used as the control. Tumor tissue slices from MDA-PATX121 were embedded with paraffin on day 0, day 3, and day 5, and tissue sections were stained with haematoxylin and eosin, and antibodies to the proliferation marker Ki-67, the fibroblast cell marker, α-SMA, endothelial cell marker, CD34, and Mason's Trichrome staining for stroma as indicated (H&E and Ki67, 10X magnification, Bars = 100 μm; αSMA and CD34, 40X magnification, Bars = 20 μm, Mason's Trichrome staining, 20x, Bars=50 μm).

Figure 2

Pathway signaling can be targeted in ex vivo tissue slice culture. (A) Tissue slices were treated with the AKT inhibitor, MK2206 (3 μM), or the MEK inhibitor, AZD6244 (3 μM), for 24 hours, and protein lysates were prepared with RIPA buffer and subjected to western blotting with the indicated antibodies. (B) Tissue slices were treated with the indicated agents for 72 hours, and tissue slice viability was measured with PrestoBlue reagent. Auranofin was used as a positive control. (C-D) Tissue slices were treated with the AKT inhibitor, MK2206 (3 μM), or the MEK inhibitor, AZD6244 (3 μM), for 24 hours, and immunohistochemical staining was performed to check p-AKT and p-ERK expression in tissue slices (200X magnification, Bars = 50 μm).

Figure 3

Computing the cut-off value to define the sensitivity or resistance of PDX tumors to chemotherapeutic agents (Gemcitabine or Irinotecan) in LTSA assay. (A) Correlation analysis of LTSA value (the ratio of fluorescence intensity of treated tissue slices over untreated control slices) with patients’ progression free survival (PFS) (GraphPad 6.0). (B) Determining the cut-off value from ROC Curve analysis. LTSA values of each PDX tumor in LTSA assay were analyzed with the program of JMP, and area under curve (AUC) values were computed by the program with manually input of various possible cut-off values. From the analysis, a cut-off LTSA value of 0.68 or 0.67 defines the borderline between sensitivity and resistance of PDX tumor in LTSA assay. (C) Patients who received LTSA sensitive regimens have significant longer PFS. Difference of PFS of patients who received LTSA resistant and sensitive regimens was analyzed using t test, and graph was made with GraphPad 6.0.

Figure 4

Responses of PDAC xenograft tumor tissue slices to therapeutic agents in the LTSA correlate with patients’ clinical responses and outcomes. (A) Tissue slices from the indicated xenografts were treated with gemcitabine or irinotecan for 72 hours, and tissue slice viabilities were measured with PrestoBlue (2 hours incubation). Significance of differences in tissue slice viabilities between treatment and control groups were analyzed with student's t-test. We defined the tissue slice as sensitive if both p<0.05 and viability was inhibited by at least 30 percent. (B) (left panel), Tissue slices from MDA-PATX106 were treated with gemcitabine or irinotecan with indicated doses for 48 hours, and protein was harvested for western blotting analysis of apoptosis markers as shown in the figures; (middle panel), after 48 hours treatment with indicated agents, tissue slices were fixed and embedded for immunohistochemical staining of Ki67 (200X magnification, Bars = 50 μm); (right panel), quantification of Ki67 staining in tissue slices with or without treatment. (C) Tissue slices from PATX106 were treated with gemcitabine or irinotecan with indicated doses for 48 hours, and western blotting (left panel) and IHC staining of Ki67 were performed (middle and right panel, image, 200X magnification, Bars = 50μ). (D) The patient, from whom the PATX106 tumorgraft was derived, responded to gemcitabine treatment as neoadjuvant and adjuvant therapy. Patient's CT images were retrieved from clinical stations and compared before and after treatments.

Figure 5

Validation of LTSA result with PDX model in vivo. PATX76 Tumorgrafts were re-grown in nude mice, and tumor bearing mice were treated with PBS or Irinotecan with 50mg/kg weekly for 3 weeks. Tumor volume were measured weekly. Mann-Whitney U test was used for significance between two treatment groups (two tailed), **, p<0.01. (A) tumors at the end point of experiment; (B) tumor growth curve. (C) Immunohistochemical staining of Ki67 in tumor tissue treated with irinotecan or PBS control (200X magnification, Bars = 50μm). (D) Quantification of Ki67 staining; values are means of positive staining number in 5 random views, t test was used for significance between two group, **, p<0.01.