Posted in: information

Tumor therapy:systematic evaluation of in vitro organoid culture system using a variety of sequencing techniques

Colorectal cancer is one of the most common cancers, and its mortality is the second highest in the world. At present,The organoid model constructed from tumor tissue of patients has become a common research method to study the molecular mechanism of tumorigenesis。 A variety of tumor like organs have been successfully constructed, including colorectal cancer, breast cancer, bladder cancer, prostate cancer, gastric cancer and so on. However, most of these studies only evaluated various molecular characteristics of patient tissue-derived organoids at the average level of a large number of cell ensembles, such as gene mutation, genomic copy number variation and gene expression. They did not evaluate at the single-cell level, so they could not systematically evaluate the tumor cell heterogeneity within the constructed organoids, especially because the success rate of constructing organoids is often not very high, At the same time, it is very difficult to obtain the tumor single-cell omics data of the same patient in vivo and the paired single-cell omics data after the establishment of organoids in vitro.

In order to systematically evaluate the organoid culture system of colorectal cancer, analyze the similarities and differences between organoids and corresponding tumor epithelial cells in vivo, and the effects of different culture systems on the expression characteristics of organoid genesBeijing Biomedical Innovation Center (biopic)Tang FujuProfessor team and general surgery of the Third Hospital of Peking UniversityFu WeiProfessor teamwork, high-precision single-cell transcriptome sequencing was performed on tumor tissues and adjacent normal tissues from 6 patients with colorectal cancer, as well as their corresponding organoids cultured in vitroWhole genome methylation sequencing, whole genome sequencing, whole exome sequencing and target Sanger sequencingThe two common colorectal organoid culture systems were systematically compared and evaluated from the three levels of transcriptome, genome and DNA methylation group (Fig. 1). The research results were published on April 28, 2022“Systematic evaluation of colorectal cancer organoid system by single-cell RNA-Seq analysis”Published online on Genome Biology Come on.

Fig. 1 Schematic diagram of experimental design scheme

The study has the following three main findings:

1. Organoids derived from tumor tissue can accurately reflect the key characteristics of tumor epithelial cells (cancer cells) in gene expression, gene mutation and DNA methylation.

By exploring the tumor specific gene expression pattern in vivo, this study found that tumor derived organoids highly expressed genes specifically expressed by tumor epithelial cells (cancer cells) in vivo. Moreover, tumor tissue-derived organoids also maintain the characteristics of tumor epithelial cell specific gene regulatory network in vivo. In addition, by analyzing the data of whole genome (WGS), whole exome (WES) and DNA methylation group (PBAT), the study found that tumor like organs can very well maintain the key characteristics of tumor epithelial cells (cancer cells) in gene mutation and DNA methylation (Fig. 2). This shows that the colorectal cancer tumor like organs in the existing culture system maintain the key biological characteristics of the cancer cells in the corresponding patients very well. Therefore, the cancer treatment candidate drugs screened by tumor like organs should have a similar killing effect on the cancer cells in the corresponding patients. The existing tumor organoids are an excellent platform for the screening of tumor killing drugs.

Fig. 2 Comparison of genomic copy number variation, DNA methylation and gene mutation between tumor cells and normal intestinal epithelial cells in vivo and in vitro

2. Organoids derived from normal tissues adjacent to cancer show some tumor like characteristics at the transcriptome level, but maintain normal genomic and global DNA methylation characteristics.

Firstly, this study identified the differentially expressed genes in tumor epithelial cells and adjacent normal intestinal epithelial cells in vivo, and studied the expression of these differentially expressed genes in tumor like organs and normal intestinal epithelial organs in vitro. The results showed that the genes specifically expressed by tumor epithelial cells in vivo were highly expressed in normal intestinal epithelial organs and tumor organs in vitro. In order to further verify this result, immunofluorescence staining of tumor specific expression gene CEACAM6 was carried out in vivo and in vitro. Consistent with the single-cell transcriptome data, CEACAM6 was highly expressed only in tumor epithelial cells in vivo, but not in normal intestinal epithelial cells adjacent to cancer in vivo. However, CEACAM6 protein was highly expressed in tumor organoids and normal intestinal epithelial organoids cultured in vitro, which was consistent with the results of single-cell transcriptome sequencing (Fig. 3). The normal tissues adjacent to the cancer in this study are taken from the area at least 10 cm away from the edge of the tumor tissue. The possibility of a large number of tumor cells mixed in the normal tissue is very low. However, in order to further exclude the normal tissue, the organoids may be caused by the expansion of tumor cells mixed in the normal tissues adjacent to the cancer in vitro, This study further explored the gene mutation and genomic copy number variation of tumor like organs and normal intestinal epithelial organs in vitro. The results showed that only tumor organoids showed genomic copy number variation and gene mutation similar to those of tumor cells in corresponding patients, while the genome of normal intestinal epithelial organoids was consistent with that of normal intestinal epithelial cells in vivo, and there were no tumor cell specific gene mutation and genomic copy number variation, which ruled out the possibility that normal tissue organoids originated from mixed tumor epithelial cells in adjacent normal tissues. These data show that both tumor epithelial organs and normal intestinal epithelial organs show tumor like characteristics at the transcriptome and protein levels. This shows that the existing culture system makes the normal intestinal epithelial organoids also have some tumor cell characteristics. Therefore, it is impossible to screen the candidate drugs with specific selective killing effect on tumor cells (killing tumor organoids but not normal intestinal epithelial organoids) by using the tumor organoids obtained from the same patient and the adjacent normal intestinal epithelial organoids.

Fig. 3 fluorescence results of CEACAM6 immunostaining

3. Conditioned medium is superior to chemical composition determining medium (molecular medium) in the long-term culture of tumor like organs.

First, the study evaluated cell proliferation in different media by the expression of mki67 (Fig. 4). In the chemical composition determination medium (molecular medium), the organoids from normal tissue have a faster cell proliferation rate than those from tumor. In the conditioned medium, the proliferation rate of normal tissue derived organoids and tumor derived organoids was the same. This shows that the chemical composition determination medium (molecular medium) is more conducive to the growth of normal intestinal epithelial cells, while the conditioned medium has no obvious preference for the growth of normal intestinal epithelial cells and tumor epithelial cells (cancer cells). This feature was further verified by the lineage tracing of cancer cells cultured in different media with mitochondrial mutation.

Figure 4 proportion of organ like cells expressing mki67 in vivo and in vitro

In addition, the results show that the conditioned medium can more truly reflect the difference between tumor epithelial cells (cancer cells) and normal intestinal epithelial cells in vivo than the chemical composition determination medium (molecular medium). Similar to the corresponding cancer cells in vivo, tumor organoids cultured in conditioned medium showed the characteristics of high expression of intestinal epithelial stem progenitor cell marker gene olfm4 and low expression of intestinal epithelial differentiation and maturation marker gene Ca2, while normal tissue organoids showed the opposite pattern of low expression of olfm4 and high expression of Ca2 (Fig. 5). However, in the chemical composition determination medium (molecular medium), both normal tissues and tumor organs have similar patterns of high expression of olfm4 and low expression of Ca2, which can not accurately simulate the different characteristics of gene expression of different types of epithelial cells in vivo. This shows that the two existing culture systems are effective in maintaining the key biological characteristics of tumor cells in vivo, but the conditioned medium is better than the chemical composition determination medium (molecular medium), so the conditioned medium is the preferred culture system for the study of molecular mechanism of tumorigenesis and drug screening based on organoids.

Fig. 5 expression of Ca2 and olfm4 in tumor cells and normal epithelial cells under different conditions

To sum up, this study conducted high-precision single-cell transcriptome analysis of tumor tissues in colorectal cancer, adjacent normal tissues in colorectal cancer, and paired tumor like organs and adjacent normal tissues and organs established in two different culture systems, combined with the results of whole genome sequencing, whole exome sequencing, DNA methylation group sequencing and target Sanger sequencing, The reliability and limitations of organoid model in studying the molecular mechanism of colorectal cancer tumorigenesis were systematically evaluated.The study found that the tumor like organs in the existing culture system well maintained the key biological characteristics of cancer cells in patients with colorectal cancer,The existing tumor organoids are a superior platform for the study of molecular mechanisms of tumorigenesis and the screening of tumor killing drugs. The existing culture system makes the normal intestinal epithelial organs also have some tumor cell characteristics, so it is impossible to pair and screen the tumor organs and normal intestinal epithelial organs obtained from the same patient, so as to obtain candidate drugs with selective killing effect on tumor cells. The two existing culture systems are effective in maintaining the key biological characteristics of colorectal cancer tumor cells, but the conditioned medium is better than the chemical composition determination medium (molecular medium)Conditioned medium is the preferred culture system for the study of molecular mechanism of colorectal cancer tumorigenesis and drug screening