Patient Derived Xenografts Expand Human Primary Pancreatic Tumor Tissue Availability for ex vivo Irreversible Electroporation Testing

Abstract

New methods of tumor ablation have shown exciting efficacy in pre-clinical models but often demonstrate limited success in the clinic. Due to a lack of quality or quantity in primary malignant tissue specimens, therapeutic development and optimization studies are typically conducted on healthy tissue or cell-line derived rodent tumors that don't allow for high resolution modeling of mechanical, chemical, and biological properties. These surrogates do not accurately recapitulate many critical components of the tumor microenvironment that can impact in situ treatment success. Here, we propose utilizing patient-derived xenograft (PDX) models to propagate clinically relevant tumor specimens for the optimization and development of novel tumor ablation modalities. Specimens from three individual pancreatic ductal adenocarcinoma (PDAC) patients were utilized to generate PDX models. This process generated 15-18 tumors that were allowed to expand to 1.5 cm in diameter over the course of 50-70 days. The PDX tumors were morphologically and pathologically identical to primary tumor tissue. Likewise, the PDX tumors were also found to be physiologically superior to other in vitro and ex vivo models based on immortalized cell lines. We utilized the PDX tumors to refine and optimize irreversible electroporation (IRE) treatment parameters. IRE, a novel, non-thermal tumor ablation modality, is being evaluated in a diverse range of cancer clinical trials including pancreatic cancer. The PDX tumors were compared against either Pan02 mouse derived tumors or resected tissue from human PDAC patients. The PDX tumors demonstrated similar changes in electrical conductivity and Joule heating following IRE treatment. Computational modeling revealed a high similarity in the predicted ablation size of the PDX tumors that closely correlate with the data generated with the primary human pancreatic tumor tissue. Gene expression analysis revealed that IRE treatment resulted in an increase in biological pathway signaling associated with interferon gamma signaling, necrosis and mitochondria dysfunction, suggesting potential co-therapy targets. Together, these findings highlight the utility of the PDX system in tumor ablation modeling for IRE and increasing clinical application efficacy. It is also feasible that the use of PDX models will significantly benefit other ablation modality testing beyond IRE.

Keywords: IRE; PDX; ablation; conductivity; inflammation; irreversible electroporation; pancreatic cancer.

Figure 1

PDX models expand small tumor specimens for ablation testing. (A) Schematic of pancreatic cancer patient-derived xenograft model. Primary human pancreatic tumor tissue was implanted into an NSG (Passage 1) and allowed to progress, excised, and expanded into larger cohort of mice (Passage 2), and then collected for histological assessment and ex vivo testing. (B) Tumor growth curve from PDX model mice. SEM, n = 16–18 mice for each model. (C) Average maximum specimen size, SEM, n = 13–16.

Figure 2

PDX tumors better recapitulate human PDAC histopathology and complexity compared to cell line based Pan02 models. (A) Representative image of PDX pancreatic tumor taken from Patient 1. Tumors from the PDX model exhibit similar characteristics as human patient samples including the formation of ductular structures and a similar tumor cell morphology. They have an identifiable collagenous stroma but not as robust as in patient samples. Mitotic figures are often abnormal. (B) Representative primary human PDAC tumor. Tumor cells from human patients often form ductular structures with lumens. Moderate to large amounts of fibrous connective tissue stroma separates tumor cells. Individual tumor cells are irregularly round with abundant amounts of eosinophilic cytoplasm and irregularly round nuclei. Mitotic figures are often abnormal. (C) Representative image of a Pano2 tumor expanded in a NSG mouse. Tumors derived from Pan02 cells lack the formation of ducts. Tumor cells are arranged in vague streams. They are more spindle in shape with less cytoplasm and elongated nuclei. The tumor stroma is scant. Mitotic figures are of normal morphology. Short arrows indicate mitotic figures, asterisks indicate ductal structures, and long arrows indicate elongated nuclei, and arrowhead indicates tumor stroma tissue. All images are HE stain and were taken at 40x magnification.

Figure 3

PDX and primary human tissues exhibit similar conductivity and temperature changes during IRE application. (A) Schematic of IRE treatment and tissue characteristic assessment ex vivo. (B) Raw and (C) percent change in conductivity for PDX (n = 3–6), primary human tissues (n = 2–3), and Pan02 tumor tissue (n = 1) for each electric field were collected at electric field magnitudes ranging from 0 to 3000 V/cm during IRE application. Conductivity values were calculated based on the average current recorded during the last 5 μs of the first IRE pulse. SEM, 2-way ANOVA (Pan02 not considered due to low n-value), p-value * < 0.05, *** < 0.001. Change in temperature induced by IRE at (D) 1,000 V/cm and (E) 2,500 V/cm were recorded throughout pulse application.

Figure 4

Modeling of PDX and primary human tissues results in similar predicted ablation sizes and damage contributions. (A) COMSOL model depiction of the predicted ablation area at 500 V/cm (gray) and thermal damage area at Ω = 1.0 (red) resulting from 100 pulses, 100 μs on time, and 1,750 V/cm voltage to distance ratio modeling clinical IRE. (B) Quantification of COMSOL model of predicted contributions of IRE and thermal damage to tumor ablation model.

Figure 5

IRE induces patient- and dose-dependent gene expression changes in PDX pancreatic tumors. (A) Gene expression arrays were utilized to evaluate changes in the expression of genes associated with cancer and cell death following IRE treatments at 0, 500, and 2,500 V/cm. A heatmap of the expression data was generated (z-score ranking ±3). N = 3–6 specimens in each group. (B) Summary table of dominate biological pathways affected by IRE in pancreatic tumor tissue from human PDX samples and from murine Pan02 samples. (C) IRE significantly alters cancer hallmark and immunosuppressive biological pathways in PDX pancreatic tumor models. IPA analysis of affected biological pathways assigned Z-scores based on predicted impact from individual gene expression changes. 0, 500, and 2500V/cm IRE treated tissues were compared and showed significantly increased necrosis, regeneration/repair, and inflammation signaling. Diagram of dose-dependent effect of IRE on biological pathways involved in cancer, cell death, and inflammation.

Figure 6

IRE treatment potently attenuated KRAS and EGFR signaling and increases antigen presentation. (A) Specific pancreatic cancer-related pathways were analyzed via IPA and showed significant alternations in gene expression of KRAS and EGFR pathway signaling molecules following IRE treatment at 2500 V/cm. n = 3–6 specimens in each group. (B) Antigen presentation pathways are significantly increased in PDX tumors following IRE treatment. Twenty-five genes were identified as being key regulators associated with the increase in antigen presentation signaling following IRE treatment (red is up-regulated; green is down-regulated). These genes are predicted to impact the function of 9 key drivers of antigen presentation and impact the biological functions shown at the bottom of the schematic, all predicted to result in increased antigen presentation.