Fig 2: Establishment of neuroendocrine neoplasm (NEN) patient-derived xenograft (PDX) models: (A) Tumor samples from NEN patients were cryopreserved, thawed, and injected into the flank of immunocompromised NOD Scid Gamma (NSG) mice. Two mice developed subcutaneous tumors at three months post injection (NEC913 and NEC1452 PDX models). Both PDX models have been passaged in 6 generations of mice. (B) Formalin-fixed and paraffin-embedded tumor sections are stained with H&E and stained for Ki67 and neuroendocrine tumor markers such chromogranin A (CgA), synaptophysin (SYP), and somatostatin receptor 2 (SSTR2) by IHC. Scale bar represents 40 µm. (C) Exome sequencing of NEC913 and NEC1452 PDX tumors demonstrated mutations in TP53 and RB1.

Fig 3: Analysis of human primary, relapsed and bone metastatic PCa specimens for the expression of CD44 and Twist1.(A) IHC staining was performed on ten primary, ten relapsed and ten bone metastatic PCa specimens using a specific antibody against a NE marker CgA. Representative images of CgA expression are shown depicting negative staining in primary and positive staining in relapsed and bone metastatic PCa. (B) The RNA level of CD44 and Twist1 were measured in each specimen by RT-qPCR. The statistical significance was calculated between the three groups of specimens using Student’s t-test. Statistical significance between groups is indicated. Data represent means ± SD. *p < 0.05 by Student’s-t test.

Fig 4: IHC analyses of NEC913 patient sample: (A) Primary NEC tumor at the ampulla of Vater. Scale bar represents 3 mm. (B) H&E staining of primary NEC tumor. (C–I) Staining for Ki67, CgA, SYP, SSTR2, ASCL1, p53, and Rb. Scale bar represents 200 µm.

Fig 5: Characterization of NEC913 and NEC1452 cells for NET markers: (A) NEC913 and NEC1452 cells incubated with antibodies against CgA (1/300), SSTR2 (1/300), and SYP (1/600) overnight and with secondary antibodies coupled to FITC (1/500) for 1 h at room temperature. Microscopy pictures are taken using 200 ms exposure time. Scale bar represents 100 µm. (B–H) Gene expression analyses of NET markers in NEC913 and NEC1452 cells compared to BON cells. Gene expression levels were normalized to the control gene GAPDH to determine the relative fold change. Statistical analyses of gene expression changes were performed using T-tests in Prism GraphPad. p < 0.05 was depicted with *. p < 0.01 was depicted with **.