Patient-Derived Organoid Models of Human Neuroendocrine Carcinoma

Krijn K Dijkstra, José G van den Berg, Fleur Weeber, Joris van de Haar, Arno Velds, Sovann Kaing, Dennis D G C Peters, Ferry A L M Eskens, Derk-Jan A de Groot, Margot E T Tesselaar, Emile E Voest, Krijn K Dijkstra, José G van den Berg, Fleur Weeber, Joris van de Haar, Arno Velds, Sovann Kaing, Dennis D G C Peters, Ferry A L M Eskens, Derk-Jan A de Groot, Margot E T Tesselaar, Emile E Voest

Abstract

Gastroenteropancreatic neuroendocrine carcinoma (GEP-NEC) is a poorly understood disease with limited treatment options. A better understanding of this disease would greatly benefit from the availability of representative preclinical models. Here, we present the potential of tumor organoids, three-dimensional cultures of tumor cells, to model GEP-NEC. We established three GEP-NEC organoid lines, originating from the stomach and colon, and characterized them using DNA sequencing and immunohistochemistry. Organoids largely resembled the original tumor in expression of synaptophysin, chromogranin and Ki-67. Models derived from tumors containing both neuroendocrine and non-neuroendocrine components were at risk of overgrowth by non-neuroendocrine tumor cells. Organoids were derived from patients treated with cisplatin and everolimus and for the three patients studied, organoid chemosensitivity paralleled clinical response. We demonstrate the feasibility of establishing NEC organoid lines and their potential applications. Organoid culture has the potential to greatly extend the repertoire of preclinical models for GEP-NEC, supporting drug development for this difficult-to-treat tumor type.

Keywords: disease modeling; extrapulmonary neuroendocrine carcinoma; gastroenteropancreatic neuroendocrine carcinoma (GEP-NEC); organoids; pre-clinical models.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2021 Dijkstra, van den Berg, Weeber, van de Haar, Velds, Kaing, Peters, Eskens, de Groot, Tesselaar and Voest.

Figures

Figure 1
Figure 1
Histomorphology of GEP-NEC organoid lines and matching original tumor. (A) Phase-contrast photomicrographs of GEP-NEC organoid lines. Scale bar = 100 µm. (B) Immunostainings of synaptophysin, chromogranin and Ki-67 for organoids and original tumor. P7 indicates passage 7. (C) Hematoxylin and eosin (H&E) stained slides of GEP-NEC organoid lines and original tumors. NEC-01013: passage (P) 12; NEC-02002: P20; NEC-02007: P3.
Figure 2
Figure 2
Mismatch repair status of GEP-NEC organoid lines. Staining of slides from NEC-01010 (P7 and P11), NEC-01013 (P13), and NEC-02002 organoids (P15), as well as the original tumor of NEC-02002, for mismatch repair proteins MLH1, PMS2, MSH2, and MSH6. Note 100% nuclear expression of MHS2 and MSH6 but absence of nuclear staining of MLH1 and PMS2 in both organoids and original tumor of NEC-02002. The brown halo on the cell border is a specific staining caused by residual Geltrex in organoid slides. Scale bar = 100 µm.
Figure 3
Figure 3
Copy number profiles of GEP-NEC organoids. (A) Copy number profiles of GEP-NEC organoids are generated based on low-coverage whole genome sequencing and represented on a log2 scale. NEC-01013: passage (P) 11; NEC-02002: P5. (B) Copy number profiles were based on SNPs identified by whole exome sequencing and analyzed by Sequenza. Allele frequencies are given as ratios and used to infer copy number. NEC-01013: P11; NEC-02002: P15.
Figure 4
Figure 4
Drug response of GEP-NEC organoids. (A, B) Viability (A) or growth-rate corrected viability (B) of cisplatin-treated GEP-NEC organoids. n = 3–4. NEC-01010: passage (P) 6-14; NEC-01013: P10-19; NEC-02002: P6-16. (C, D) Viability (C) or growth-rate corrected viability (D) of everolimus-treated GEP-NEC organoids. n = 3–5. NEC-01010: P6-16; NEC-01013: P10-20; NEC-02002: P6-17. (E) GR at 25 nM everolimus (corresponding to Cmax). n = 3–5. Error bars represent s.e.m. Student’s t test. *P < 0.05; **P < 0.01.

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