Real time ex vivo chemosensitivity assay for pancreatic adenocarcinoma.

BACKGROUND: Patient-derived organoids (PDOs) and xenografts (PDXs) have been extensively studied for drug-screening. However, their usage is limited due to lengthy establishment time, high engraftment failure rates and different tumor microenvironment from original tumors. To overcome the limitations, we developed real time-live tissue sensitivity assay (RT-LTSA) using fresh tumor samples. METHODS: Tissue slices from resected pancreatic cancer samples were placed in 96-well plates, and the slices were treated with chemotherapeutic agents. The correlation between the chemo-sensitivity of tissue slices and each patient's clinical outcome was analyzed. RESULTS: The viability and tumor microenvironment of the tissue slices are well-preserved over 5 days. The drug sensitivity assay results are available within 5 days after tissue collection. While all 4 patients who received RT-LTSA sensitive adjuvant regimens did not develop recurrence, 7 of 8 patients who received resistant adjuvant regimens developed recurrence. We observed significantly improved disease-free survival in the patients who received RT-LTSA sensitive adjuvant regimens (median: not reached versus 10.6 months, P = 0.02) compared with the patient who received resistant regimens. A significant negative correlation between RT-LTSA value and relapse-free survival was observed (Somer's D: -0.58; P = 0.016). CONCLUSIONS: RT-LTSA which maintains the tumor microenvironment and architecture as found in patients may reflect clinical outcome and could be used as a personalized strategy for pancreatic adenocarcinoma. Further, studies are warranted to verify the findings.

Translational relevance of SOS1 targeting for KRAS-mutant colorectal cancer.

It has been challenging to target mutant KRAS (mKRAS) in colorectal cancer (CRC) and other malignancies. Recent efforts have focused on developing inhibitors blocking molecules essential for KRAS activity. In this regard, SOS1 inhibition has arisen as an attractive approach for mKRAS CRC given its essential role as a guanine nucleotide exchange factor for this GTPase. Here, we demonstrated the translational value of SOS1 blockade in mKRAS CRC. We used CRC patient-derived organoids (PDOs) as preclinical models to evaluate their sensitivity to SOS1 inhibitor BI3406. A combination of in silico analyses and wet lab techniques was utilized to define potential predictive markers for SOS1 sensitivity and potential mechanisms of resistance in CRC. RNA-seq analysis of CRC PDOs revealed two groups of CRC PDOs with differential sensitivities to SOS1 inhibitor BI3406. The resistant group was enriched in gene sets involving cholesterol homeostasis, epithelial-mesenchymal transition, and TNF-α/NFκB signaling. Expression analysis identified a significant correlation between SOS1 and SOS2 mRNA levels (Spearman's ρ 0.56, p < 0.001). SOS1/2 protein expression was universally present with heterogeneous patterns in CRC cells but only minimal to none in surrounding nonmalignant cells. Only SOS1 protein expression was associated with worse survival in patients with RAS/RAF mutant CRC (p = 0.04). We also found that SOS1/SOS2 protein expression ratio >1 by immunohistochemistry (p = 0.03) instead of KRAS mutation (p = 1) was a better predictive marker to BI3406 sensitivity of CRC PDOs, concordant with the significant positive correlation between SOS1/SOS2 protein expression ratio and SOS1 dependency. Finally, we showed that GTP-bound RAS level underwent rebound even in BI3406-sensitive PDOs with no change of KRAS downstream effector genes, thus suggesting upregulation of guanine nucleotide exchange factor as potential cellular adaptation mechanisms to SOS1 inhibition. Taken together, our results show that high SOS1/SOS2 protein expression ratio predicts sensitivity to SOS1 inhibition and support further clinical development of SOS1-targeting agents in CRC.

Establishing a living biobank of patient-derived organoids of intraductal papillary mucinous neoplasms of the pancreas.

Pancreatic cancer (PaCa) is the third leading cause of cancer-related deaths in the United States. There is an unmet need to develop strategies to detect PaCa at an early, operable stage and prevent its progression. Intraductal papillary mucinous neoplasms (IPMNs) are cystic PaCa precursors that comprise nearly 50% of pancreatic cysts detected incidentally via cross-sectional imaging. Since IPMNs can progress from low- and moderate-grade dysplasia to high-grade dysplasia and invasion, the study of these lesions offers a prime opportunity to develop early detection and prevention strategies. Organoids are an ideal preclinical platform to study IPMNs, and the objective of the current investigation was to establish a living biobank of patient-derived organoids (PDO) from IPMNs. IPMN tumors and adjacent normal pancreatic tissues were successfully harvested from 15 patients with IPMNs undergoing pancreatic surgical resection at Moffitt Cancer Center & Research Institute (Tampa, FL) between May of 2017 and March of 2019. Organoid cultures were also generated from cryopreserved tissues. Organoid count and size were determined over time by both Image-Pro Premier 3D Version 9.1 digital platform and Matlab application of a Circular Hough Transform algorithm, and histologic and genomic characterization of a subset of the organoids was performed using immunohistochemistry and targeted sequencing, respectively. The success rates for organoid generation from IPMN tumor and adjacent normal pancreatic tissues were 81% and 87%, respectively. IPMN organoids derived from different epithelial subtypes showed different morphologies in vitro, and organoids recapitulated histologic and genomic characteristics of the parental IPMN tumor. In summary, this preclinical model has the potential to provide new opportunities to unveil mechanisms of IPMN progression to invasion and to shed insight into novel biomarkers for early detection and targets for chemoprevention.