Organoids and metastatic orthotopic mouse model for mismatch repair-deficient colorectal cancer

Abstract

Background: Genome integrity is essential for the survival of an organism. DNA mismatch repair (MMR) genes (e.g., MLH1, MSH2, MSH6, and PMS2) play a critical role in the DNA damage response pathway for genome integrity maintenance. Germline mutations of MMR genes can lead to Lynch syndrome or constitutional mismatch repair deficiency syndrome, resulting in an increased lifetime risk of developing cancer characterized by high microsatellite instability (MSI-H) and high mutation burden. Although immunotherapy has been approved for MMR-deficient (MMRd) cancer patients, the overall response rate needs to be improved and other management options are needed.

Methods: To better understand the biology of MMRd cancers, elucidate the resistance mechanisms to immune modulation, and develop vaccines and therapeutic testing platforms for this high-risk population, we generated organoids and an orthotopic mouse model from intestine tumors developed in a Msh2-deficient mouse model, and followed with a detailed characterization.

Results: The organoids were shown to be of epithelial origin with stem cell features, to have a high frameshift mutation frequency with MSI-H and chromosome instability, and intra- and inter-tumor heterogeneity. An orthotopic model using intra-cecal implantation of tumor fragments derived from organoids showed progressive tumor growth, resulting in the development of adenocarcinomas mixed with mucinous features and distant metastasis in liver and lymph node.

Conclusions: The established organoids with characteristics of MSI-H cancers can be used to study MMRd cancer biology. The orthotopic model, with its distant metastasis and expressing frameshift peptides, is suitable for evaluating the efficacy of neoantigen-based vaccines or anticancer drugs in combination with other therapies.

Keywords: Lynch syndrome; MSH2; chromosome instability; colorectal cancer; microsatellite instability; mismatch repair deficiency; mouse model; organoid.

Figure 1

Characterization of organoid line 586T2A4 by (A) IHC staining, (B) mRNA in situ hybridization, and (C) TUNEL staining. Scale bar: 50 µm.

Figure 2

Fragment analysis of seven MSI markers in 586T2A4-P8-T11 and WT organoids. Blue peaks are 586T2A4-P8-T11 and WT organoids, pink or green peaks are WT tail, and yellow peaks are size markers. Two vertical red lines align the WT fragment peaks in WT tail.

Figure 3

Karyotype by SKY analysis in (A) 586T2A4: 40,XY,+1,-2,+3,T(3;4),-8,+9(T6),-10,+12,-15,-16. T: translocation. One copy of chromosome 15 was bigger than that in (B) a WT normal cell.

Figure 4

Asxl1 expression by RNA in situ hybridization in organoids 586T2A4 and 961T3C3. Two RNAScope® probes were designed upstream (red; left panel) and downstream (yellow; the panel next to the left) of the mononucleotide repeat region in Asxl1 mRNA. 3rd panel: merged images of two probes; right panel: merged images of two probes and DAPI for nuclei counter staining. WT: both yellow and red signals (merged as orange, red arrow). Mutant: red only (blue arrow). Scale bar: 50 µm.

Figure 5

In vivo growth of organoids. (A) Growth of parental organoids 586T2A4 and 961T3C3 by s.c. injection of 1 × 10⁶ cells in C57BL/6J recipients (n = 10 per group). This was in vivo passage 0 (P0). *Tumor fragments from P0 tumor #3268 (left panel) and #3274 (right panel) were serially passaged s.c. in vivo. (B) Growth of 586T2A4 serially passaged tumor fragments at P9 from two different donor tumors (n = 5 per group). The median time for a tumor to reach 500 mm³ was 26.4 days (95% CI, 21.5 to 31.3) (left panel) and 28.3 days (95% CI, 26.4 to 35) (right panel). TV, tumor volume.

Figure 6

Intra-cecal implantation model developed using P8 tumor fragments from 586T2A4 organoids (n = 5 per group). (A) Kaplan-Meier survival curve using tumor fragments from three different donor tumors originating from 586T2A4 organoids. The median survival was 60, 67, and 78 days post implantation (dpi). (B) Gross primary cecal tumor and liver metastasis (red arrows). (C) H&E staining of cecal tumors and liver metastasis. The red star indicates the invasion of tumors into intestinal serosa. Scale bar: 200 µm (left panel) and 25 µm (middle and right panels). (D) H&E staining of lymph node metastasis. Scale bar: 50 µm. (E) Images of ultrasound (US; left) and MRI (right, red arrow points to tumor mass). (F) Tumor volume (TV) measured by MRI.