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. Author manuscript; available in PMC: 2017 Dec 15.
Published in final edited form as: Clin Cancer Res. 2016 Jun 3;22(24):6021–6030. doi: 10.1158/1078-0432.CCR-15-2936

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

David Roife 1,#, Bingbing Dai 1,¶,#, Ya'an Kang 1, Mayrim V Rios Perez 1, Michael Pratt 1, Xinqun Li 1, Jason B Fleming 1,
PMCID: PMC5136340  NIHMSID: NIHMS793631  PMID: 27259561

Abstract

Purpose

Translation of the 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 3-5 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 pancreatic cancer patients.

Keywords: Pancreatic Cancer, Patients-Derived Xenografts, Ex Vivo Tissue Slice Cultures

Introduction

Biomarker-guided cancer therapy has improved patient outcomes, but predictive biomarkers are available in only a small subset of the patients (1). Therefore, new approaches to personalized cancer therapy are necessary to match individual tumors with effective agents. Evidence from recent studies has demonstrated that tumorgrafts, or patient-derived xenografts (PDX), established by engrafting patient tumors into immunocompromised mice, preserve the structural components, heterogeneity, gene expression pattern, and genetic mutations of the original tumors (2-4). These features are maintained through several generational passages of the tumorgrafts in mice (5, 6). Direct tumorgrafts have also been shown to predict clinical responses of developing drugs (7-10) and guide clinical treatment in selected case studies (11-14). However, the major hurdle for applying the PDX model to personalized medicine is that establishing large-scale tumorgrafts is expensive and time-consuming (10, 15).

Recently, ex vivo cultures of tumor tissue slices derived from prostate, renal cell carcinoma, head and neck, and breast cancers have been established with the aim to study the tumor biology and test drug activities (16-24). Because tissue slices in ex vivo culture keep some components of the tumor microenvironment, they are believed to have similar predictability as in vivo tumor models. In addition, tissue slices can be cultured and treated with therapeutic agents similarly to traditional cell line-based assays, thus significantly reducing the time for drug testing compared to animal models. However, previously reported tissue slice culture assays were performed in 6-well or 24-well plates, and the evaluation of drug activity was performed by immunohistochemical staining, which is not feasible for rapid testing against multiple possible agents and drug combinations (22, 23). In this study, we report a simple, reproducible, and scalable ex vivo live tissue sensitivity assay (LTSA) that mirrors pancreatic ductal adenocarcinoma (PDAC ) patient responses to therapeutic agents. This system allows testing the sensitivity of an individual tumor against a large set of therapeutic agents in 3-5 days, and our pilot study indicates that LTSA is highly predictive of patients’ clinical responses. Recently, Majumder et al. reported another ex vivo tissue slice culture system (CANScript) in the format of 48-well plate with high predictability for patients’ responses to chemotherapeutic agents (25). Both our study and the study by Majumder et al. suggest the feasibility and reliability of using large scale ex vivo tissue slice culture systems for predicting patients’ responses to personalized cancer treatments.

Materials and Methods

Reagents and animals

Cell culture medium RPMI1640 was purchased from HyClone. Fetal bovine serum (FBS) and PrestoBlue® Cell Viability Reagent were purchased from Life Science Technologies (Grand Island, NY). Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture 100× and Auranofin were purchased from Sigma (Missouri, MO). p-AKT(Y473), p-ERK(S44/42), PARP, Cleaved-PARP, Caspase-3, Cleaved-Caspase 3, Ki-67, and β-Actin antibodies were purchased from Cell Signaling Technology (Cell Signaling, Danvers, MA). α-SMA and CD34 antibodies were purchased from Abcam (Cambridge, MA). Belzer UW® Cold Storage Solution was provided as a gift from Bridge to Life Ltd (Columbia, SC). Gemcitabine and irinotecan were provided from the pharmacy department of M.D. Anderson Cancer Center. MEK inhibitor AZD6244 and AKT inhibitor MK2206 were purchased from Selleck Chem Company (Houston, TX). NOD/SCID and Nude mice (female, 6 weeks) were purchased from the National Cancer Institute and Jackson Laboratories.

Establishment of Patient-derived Xenografts

Pancreatic cancer tumor xenografts were established from patients who have given written informed consent for our IRB-approved laboratory research protocol (LAB070854). Briefly, fresh tumor samples of about 2 mm3 were cut from patient tumor specimens, briefly soaked in Matrigel (100%), and implanted into the subcutaneous space of NOD/SCID mice, as described previously (15). The tumors were harvested when they reached 0.75-1.5 cm in diameter and labeled F1 to F5 to indicate different generational passages in animals.

Ex vivo tissue slice culture and drug treatment

Tissue cores were generated with 3-mm disposable biopsy punches (Integra Miltex, York, PA) from tumorgrafts and immediately put in Belzer UW® Cold Storage Solution supplemented with 2% Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture. Tissue cores were embedded in 1% low melting-point agarose gel (Sigma, St. Louis, MO) and cut into slices (200 μm) with the Krumdieck Tissue Slicer (Alabama Research and Development, Munford, AL). With this technique, depending on the number of cores obtained, approximately 100-150 tissue slices were generated from a single tumorgraft. The tissue slices were randomly arrayed in 96-well plates with 100 μl RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 2% PSN and incubated in a humidified 37°C incubator supplied with 5% CO2. The plates were seated on a platform shaker at 150 RPM. After 2 hours incubation, tissue slices were treated with gemcitabine (30 μM, 100 μM), irinotecan (10 μM, 30 μM), MEK inhibitor AZD6244 (3 μM), or AKT inhibitor MK2206 (3 μM) in an additional 100ul medium, totaling 200 μl medium per well. Auranofin (10 μM) was used as a positive control. The plates were returned to the incubator/shaker and cultured for 24-72 hours.

Tissue slice viability assay

After the treatment period, 20 μl of 10X Prestoblue® reagent was added to the tissue slice culture medium, and the plates were incubated for an additional 2 hours on the shaker. Tissue slice viabilities were measured through reading fluorescence intensity with a CLARIOstar® plate reader (BMG LABTECH, Cary, NC) and were normalized against the viability of control (untreated) slices.

Western blotting

Tissue slices treated with therapeutic agents for 24 to 72 hours were transferred to 1.5 ml Eppendorf tubes and washed with cold PBS, and tissue lysates were extracted with RIPA buffer and quantified for protein concentration. Thirty to fifty micrograms of protein was used for western blotting analysis. Detailed methods can be found in our previous publication (26).

Masson's trichrome staining

Paraffin embedded tumor tissue slices were prepared and cut into 5 μm sections. Masson's trichrome stain was performed according to the manufacturer's instructions (Trichrome Stain Masson Kit; Sigma-Aldrich, St. Louis, MO).

Immunohistochemistry staining

Tissue slices with or without treatment were fixed with 10% formalin for 2 hours followed by embedding in paraffin. Tumor tissues harvested from mice were fixed for overnight. Embedded tumor or tissue slices were cut into 5μm sections. Immunohistochemistry staining was performed with the Lab-Vision 480-2D immunostainer (Thermo Fisher, Fremont, CA). The staining results were visualized with diaminobenzidine as a chromogen. Images were captured with an Olympus DP72 camera and CellSens software (Center Valley, PA) on an Olympus BX51 microscope.

Animal experiment in vivo

Animal experiment protocol (00001089-RN00) was reviewed and approved by The University of Texas MD Anderson Cancer Center (Houston, Texas) institutional review board and in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health. Tumorgrafts stored in Storage Solution were recovered from liquid nitrogen and re-implanted with sizes of ~10 mm3 in nude mice. When tumors reached the sizes about 100 mm3, mice were randomly divided into 2 groups and treated with irinotecan with 50 mg/kg or PBS weekly. The tumor volumes were calculated using the formula, length × width2 × 0.52. For each group, 1 mouse was euthanized after the 2 week (14 days) treatment. Tumors were harvested for histochemical analysis. When tumors reached the size of 12 mm in diameter, all mice were sacrificed, and tumors were harvested for histochemical analysis.

Statistical analysis

The significance of differences in tissue slice viabilities between treatment and no-treatment groups was analyzed by Student t-test (two tails). Cut-off value for defining the sensitivity in LTSA assay was determined with ROC curve analysis. LTSA values of PDX tumors in LTSA assay were analyzed with the JMP software (SAS institute), and area under curve (AUC) values were computed by the program with manually input of various possible cut-off values. Correlation of LTSA results (sensitive or resistance) with patients PFS were also analyzed with Pearson correlation analysis. The significance of the in vivo animal study data was determined by Mann-Whitney U test (two tailed) with GraphPad Prism 6.0.

Results

Establishment of live tissue sensitive assay (LTSA)

The aim of this study is to develop an ex vivo method to test the drug activity in live tumor tissue from mice or patients with high efficient fashion and to test its clinical relevance. With a microtome machine we can produce precision cut tumor tissue slices with uniform sizes and measure drug activities in a 96-well plate within 24-72 hours. The flowchart of this method is shown in Figure 1A: When tumorgrafts reach ~1 cm in diameter, a size without tissue necrosis, tumors are harvested for tissue slice processing. Tissue cores are taken using a core biopsy punch (3 mm) from PDX tumor and embedded in 1% agarose gel, and then the tissue cores are cut with a microtome tissue slice cutter into tissue slices with a thickness of 200 μm. The tissue slices are manually arrayed in 96-well plates with 100 μl cell culture medium and treated with therapeutic agent. Here, we used a testing agent, auranofin, as an example. After treatment with 3 different doses of auranofin for 72 hours, tissue slices viabilities were measured with PrestoBlue®. The PrestoBlue® reagents changed color to purple after 2 hours incubation. Inhibition of tissue viability by therapeutic agent are calculated through normalization of the fluorescence value of treated slices by that of control slices. As was shown in figure 1A (last panel), viability of MDA-PATX137 tissue slices were inhibited by auranofin with dose-dependent manner. PrestoBlue® is a metabolic-based cell viability reagent, and it has the advantage of allowing direct reading tissue slice viability without removing culture medium. With this technique, 100-150 tissue slices can be produced from a single tumorgraft and used for testing the responses of xenografts to therapeutic agents within 3 days.

Figure 1.

Figure 1

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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).

Ex vivo tissue slice culture maintains the microenvironment and structure of tumorgrafts

Previous studies showed that tissue slices can remain viable for at least 4 days in 6- or 24-well plate culture (27). To test the viabilities of the tissue slices over a 5-day culture period in a 96-well plate, tissue slices were produced from patient-derived xenografts MDA-PATX121 and MDA-PATX124, which were derived in our laboratory from 2 different patients’ tumors respectively. The tissue slices were cultured in 96-well plates with 200 μl medium in each well with no treatment. The tissue slices’ viabilities were measured with PrestoBlue every 24 hours. Tissue slices in ex vivo culture kept about 90% of the original viability for 3 days and 60%-80% viability even at day 5 (Fig. 1B). Tissue architecture such as stromal and collagen (marked by α-SMA and trichrome staining) and vascular endothelial cells (marked by CD34) were well preserved in ex vivo tissue slice culture (Fig. 1C). These results suggest that short-term ex vivo tissue slice culture in our system can adequately retain the viability, tumor structures, and microenvironment components.

Pathway signaling can be targeted in ex vivo tissue slice culture

To test whether signaling pathways in ex vivo tissue slice culture could be inhibited by targeted therapeutic agents, tissue slices were treated with an AKT inhibitor, MK2206, or an MEK inhibitor, AZD6244, for 48 hours. Western blotting was performed to check the level of p-AKT and p-ERK in the tissue slices with or without treatment. Both p-AKT and p-ERK were completely inhibited in the treated tissue slices (Fig. 2A). Although the inhibition of the targets, p-AKT and p-ERK, was not associated with viability inhibition of tissue slices (Fig. 2B); this is consistent with previous clinical studies showing that inhibition of AKT or MEK pathway alone was not significantly effective in pancreatic cancer treatment (28, 29). Immunohistochemical staining also confirmed that t p-AKT and p-ERK were inhibited by AKT inhibitor and MEK inhibitor, respectively, in tissue slices derived from both PATX135 and PATX140 tumors (Fig. 2C and 2D), indicating that targets can be specifically inhibited by therapeutic agents in the ex vivo tissue slice culture system. These observations suggest that critical biologic pathways in the pancreatic cancers remain intact during the assay which will be particularly important when testing the activity of targeted therapeutic agents.

Figure 2.

Figure 2

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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).

Ex vivo tissue slice culture-based LTSA correlates with patients’ clinical responses

To determine if the sensitivities of pancreatic tumorgrafts to conventional chemotherapeutic drugs in LTSA assay are reflective of patients’ responses to chemotherapies in the clinic, we tested PDAC-derived PDX tumors with two standard PDAC chemotherapeutics, gemcitabine (30 and 100 μM) and irinotecan (10 and 30 μM) in 18 PDX tumors derived from different PDAC patients . The responses or LTSA value of PDX tumors to gemcitabine and irinotecan were obtained through normalizing the fluorescence intensity of treated tissue slices with that of untreated control (Table 1). We found that LTSA values (from the highest concentration of chemotherapeutic agents) in the PDX tumors are reversely correlated with patients PFS (Fig.3A). We used the receiver operating characteristic (ROC) curve analysis of LTSA value from the highest dose of gemcitabine or irinotecan, and identified that a value of 0.68 is the optimal cut-off (when AUC=1) to define the sensitivity (LTSA value≤0.68) or resistance (LTSA value>0.68) of tissue slices to the treatment (Fig.3B). With this cut-off, we found that 5 PDX tumors were sensitive to gemcitabine or irinotecan treatments and 13 PDX tumors were resistance to the treatments (Table 1). In addition, retrospective analysis of the LTSA results and clinical patients’ outcome indicated that sensitivity of PDX tumors in LTSA assay is highly correlated with patients PFS (r=0.77, p=0.0002), and that patients who received LTSA sensitive regimens had significantly longer average PFS time (16.33 vs 3.8; Table 1 and Fig. 3C). These results indicate that LTSA results are in concordance with patients’ clinical responses and outcome.

Table 1.

Correlation of LTSA results with patients' clinical responses to chemotherapeutics

Systemic Rx LTSA Sensitivity LTSA value PFS (months) Average PFS (months) after Rx
Patient received LTSA-sensitive chemotherapy MDA-PATX76 (lung met) FOLFIRINOX S 0.63936 9 16.33
MDA-PATX81 FOLFIRINOX/Gem S 0.65977 11
MDA-PATX106 Gem S 0.43088 27†
MDA-PATX107 Gem S 0.66584 26
MDA-PATX141 Gem S 0.61646 11
MDA-PATX142 Gem S 0.30694 14†
Patient received LTSA-resistant chemotherapy MDA-PATX161 FOLFIRINOX/Gem R 0.68007 8 3.8
MDA-PATX97 Gem Cisplatin R 0.73359 5
MDA-PATX100 Gem R 0.84183 0
MDA-PATX104 Gem R 0.99103 0
MDA-PATX118 Gem R 0.79856 6
MDA-PATX124 Gem R 0.80463 4
MDA-PATX137 Gem R 0.75039 6
MDA-PATX136 FOLFIRINOX R 1.02023 5
MDA-PATX144 Gem R 1.06664 8
MDA-PATX140 Gem R 1.18407 4
MDA-PATX148 (liver met) Gem Abraxane R 0.94166 0*
MDA-PATX153 (liver met) Gem Abraxane R 1.00245 0*
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Note: Pearson correlation test: r=0.77; p=0.0002

R= resistant; S= Sensitive

*

Metastasis tumor

†

currently without evidence of disease.

PFS=0 months means tumor continued progression at the immediate (first) follow-up CT imaging (<2 months).

Figure 3.

Figure 3

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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.

Validation of LTSA assay results in vitro

As was shown in some examples in Figure 4, tissue slices derived from different xenografts had different responses to the chemotherapeutics. Tissue slices from the MDA-PATX76 and MDA-PATX100 tumorgrafts were susceptible to irinotecan but not gemcitabine, whereas MDA-PATX106 was sensitive to gemcitabine but not irinotecan (Fig. 4A). Consistent with the viability assay, western blotting and immunohistochemical staining further confirmed that gemcitabine but not irinotecan induced cleaved-PARP and Ki67 reduction in MDA-PATX106 tissue slices (Fig. 4B). In contrast, irinotecan induced apoptosis and Ki67 reduction in tissue slices of MDA-PATX76 (Fig. 4C). Staining of the cleaved-Caspase 3 also showed that gemcitabine and irinotecan induced dramatic increase of cleaved-Caspase 3 in LTSA sensitive tissue slices (s-Fig. 1) MDA-PATX76 was derived from a pulmonary metastasis of a pancreatic adenocarcinoma patient that had been extensively pre-treated with gemcitabine while the disease continued progressing. After the pulmonary tumor resection, the patient was switched to FOLFIRINOX, and achieved 9 months of progression-free survival. The disease progressed a few months after ceasing FOLFIRINOX therapy, and never achieved any other periods of stability (irinotecan-based therapy was not attempted again). MDAPATX106 was derived from a primary pancreatic adenocarcinoma, and the LTSA predicted susceptibility to gemcitabine but not to irinotecan. This patient responded to gemcitabine as both neoadjuvant and adjuvant treatment (Fig. 4D), and is currently cancer-free for 27 months post-resection.

Figure 4.

Figure 4

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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.

Validation of LTSA results with PDX model in vivo

To further validate the LTSA result, we chose MDA-PATX76 as an example to confirm its sensitivity to irinotecan treatment in vivo. Based on LTSA result (Fig. 4), MDAPATX76 was susceptible to irinotecan treatment. As was shown in figure 4, tumor growth of MDA-PATX76 was significantly inhibited by irinotecan treatment (Fig. 5A and B). In addition, tumor shrinkage was observed in some of the mice in the irinotecan treated group. The cell proliferation marker, Ki67 was significantly reduced in irinotecan treated tumor (Fig. 5C and D), suggesting that tumor cell proliferation was suppressed by irinotecan treatment. This result suggest that results from ex vivo culture are consistent with responses of tumorgrafts in vivo.

Figure 5.

Figure 5

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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.

Discussion

Currently, there is no reliable method to prospectively match the therapeutic regimens to individual PDAC patients. Although PDX models have been proved to be a useful approach to personalizing cancer treatment, testing the multiple therapeutic regimens using animal model in vivo usually takes longer time than patients can wait. In this study we developed a simple and ex vivo drug testing method which can be performed in a 96-well plate, thus dramatically reducing the time and resources needed to test therapeutic agents in vivo. In our method, which is termed the Live Tissue Sensitivity Assay (LTSA), the tumor tissue slices remain viable for at least 5 days, and the tumor architecture, signaling pathways, and microenvironment components are well preserved. In addition, the biologic integrity of the LTSA is supported by the fact that signaling pathways, such as AKT and MAPK, can be efficiently inhibited by targeted agents. Most importantly, we observed that clinical responses to therapeutic agents are concordant with the responses observed in LTSA assay. Our study demonstrates the feasibility of using ex vivo-based drug testing method to match current chemotherapeutics to individual PDAC patients.

Several ex vivo-based methods including patient-derived tumor material culture, circulating tumor cells, and organoids cultures have been developed and used for testing clinical therapeutic regimens, and these approaches have demonstrated early promise in personalized cancer treatments (24, 30, 31), however, most of such approaches take long time to get enough materials for drug testing and lack intact tumor microenvironmental components which have important influence on therapeutic responses (32). Organotypic cultures using 3D cultured tumor cells or live tissue slice have also been developed previously in evaluating clinical therapeutics (33-35). Most of tissue slice-based cultures and drug testing are in the format of 6-well or 24-well plate (36, 37), and tissue slice viability was measured with labor and time-intensive methods such as immunohistochemistry staining or western blotting, which are not applicable for large-scale drug testing (19, 27). In our system, the LTSA can be performed in a 96-well-plate format and allows testing drug sensitivity in 3-5 days objectively with a commercially available reagent, which will significantly reduce the time and resources compared to the test in animal models.

The tissue slices in our system maintain about 80% of their original viability for 5 days, and targets can be efficiently inhibited with targeted therapeutic agents. Immunohistochemical staining for Ki67 expression showed positive staining in tissue slices during 5 days of culture, and almost all of the Ki67 positivity was localized to the cancer cells, rather than the stroma. Although we observed loss of stromal components at day 5, testing can be completed in 2-4 days so this should not affect the overall predictive capability of this system. Another notable observation is that even treated with very high concentrations of chemotherapeutic agents, the inhibitions of tissue slice viability was rarely above 40% in the sensitive tissue slices. This could be caused by the presence of other microenvironmental components, such as endothelial cells and stromal cells which may not respond to therapeutic agents. Stromal cells and ECM components may also shield the cancer cells from the full effect of the chemotherapy (38).

Several aspects of the assay will be refined through future exploration. First, although the tumor tissue slices remain viable for at least 5 days, this could be improved through supplementation with growth factors, such as EGF or bFGF, as described in previous reports (36). Additional modification could allow the assay to more closely recapitulate the human tumor microenvironment. For example, since tumors are often under hypoxic stress, performing the assay under these conditions may improve the predictability of this system. In addition, testing tumor tissue from orthotopic PDX tumor model will more closely mimic the tumor microenvironments of human PDAC tumors, and comparisons of the LTSA results with subcutaneous and orthotopic xenograft will be performed in the future. Moreover, passaging the tumorgrafts from F1 to F5 in mice causes losing the stroma and vascular components in the tumor (3, 4), therefore early passages of tumorgrafts should be used for the testing whenever there are available. Lastly, about 30% of the implanted patients tumors grow slow or cannot form tumorgrafts in mice, in such cases, patients will not have the chance to receive the therapy defined by LTSA assay with PDX tumors, therefore, testing of patients’ tumor with LTSA immediately after surgery is our future direction of study.

Collectively, here we report the establishment of a PDX-based ex vivo live tissue sensitivity assay (LTSA), which is reliable and predictive of clinical responses and will allow additional high-throughput testing against a large set of therapeutic agents to be efficiently performed. This system could be used for predicting patient responses to certain therapeutic agents in personalized cancer medicine and for large scale drug activity testing in new drug screening and lead drug optimization. Because PDX models have been widely established with many types of cancers, our system may significantly broaden the use of PDX models in translational cancer research. In addition, this method can directly test human resected tumor tissues if enough specimens are available. Direct testing patients’ tumor tissue with LTSA will allow assessing the activities of multiple therapeutic regimens within 2-3 days post surgeries, and the clinicians can get drug response information in a timely manner. A pilot clinical trial is under planning to apply LTSA method to guiding personalized therapy for PDAC patients. We expect that this novel method could eventually be applied to personalized cancer treatment as an independent approach or in combination with molecular biomarker-guided strategies.

Supplementary Material

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Translational relevance.

In the systemic treatment of Pancreatic Ductal Adenocarcinoma, there is no reliable method to match specific treatment to each individual patient. Although patient-derived xenograft models have been proved to be effective in prioritizing therapeutic regimens in personalized cancer medicine, establishing and testing transplantable tumorgrafts is expensive and time-consuming. The ex vivo drug efficacy testing method, LTSA, developed in this study, is in the format of 96-well-plate combined with a metabolic cell viability readout, which enables high-throughput testing of PDX tumor against a large set of therapeutic agents in a short timeframe. And this system is highly reflective of patients’ responses to conventional therapeutic agents, thus having significant potential in personalized pancreatic cancer treatment design. In addition, this approach could be used for ex vivo drug testing and lead drug optimization in developing new therapies. Therefore, this study is highly translational.

Acknowledgements

We thank all the patients who participated in our PDX research program at MD Anderson Cancer Center. We also thank Dr. Yeonju Lee and Dr. Eugene Jon Koay for their helpful suggestions and discussion.

Grant support: This work was funded by Skip Viragh Foundation, the Various Donors in Pancreatic Cancer Research Fund, and the Research Animal Support Facility—Houston under NIH/NCI award (P30CA016672). This work was also supported in part by National Institutes of Health (NIH) grant T32CA009599 (DR and M.V.R.P.)

Footnotes

Conflict of interest declaration: none

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2
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