EP-3835417-B1 - METHOD FOR CULTURING CANCER TISSUE OR TISSUE ANALOGOUS TO CANCER TISSUE

Inventors

• KAWAI, SHIGETO
• YAMAZAKI, MASAKI
• NAKANO, KIYOTAKA
• SUZUKI, MASAMI
• YAMAZAKI, TATSUMI

Dates

Publication Date

20260506

Application Date

20190808

Claims (8)

1. A culture method for forming a treatment resistant organoid, wherein the method comprises a step of three-dimensionally culturing an organoid which was treated with at least one anticancer agent and isolating a treatment resistant organoid that is resistant to the treatment with said anticancer agent, wherein the step of three-dimensionally culturing is culturing in the absence of said at least one anticancer agent and wherein the organoid is formed by a pre-culture step of culturing one or more cancer stem cells.
2. The method according to claim 1, wherein the anticancer agent is one or more anticancer agents selected from agents that inhibit the growth of cancer cells.
3. The method according to claim 1 or 2, wherein the three-dimensional culture step comprises allowing the organoid which was treated with at least one anticancer agent to adhere to a first extracellular matrix and culturing by adding a first medium.
4. The method of any one of claims 1 to 3, wherein said treatment resistant organoid has at least one of the following histochemical characteristics: (a) exhibits protrusions on the tissue surface composed of monolayer to multilayer cuboidal cells; (b) is composed of large circular-oval cells containing cell debris in the organoid or lumen; (c) exhibits compartmentalization, optionally wherein new glandular structures are formed; and (d) is CD44 positive.
5. The method according to any one of claims 1 to 4, further comprising analyzing the gene expression level, protein expression level, protein modification, protein localization, epigenome, or metabolomics state of a cell comprised in the treatment resistant organoid.
6. The method according to any one of claims 1 to 4, comprising identifying an agent having a therapeutic activity against cancer, wherein the method further comprises: (a) selecting at least one test agent; (b) contacting the treatment resistant organoid with the test agent; and (c) measuring the cytotoxic activity of the test agent against a cell comprised in the treatment resistant organoid or comparing the protein expression of a cell comprised in the treatment resistant organoid in the presence and absence of the agent.
7. The method according to claim 6 which is for identifying the mechanism of action of said agent having a therapeutic activity against cancer, wherein step (c) is comparing the protein expression of a cell comprised in the treatment resistant organoid in the presence and absence of the agent.
8. The method according to claim 6 which is for identifying a combination for combined use of an anticancer agent and said agent having a therapeutic activity against cancer, wherein step (c) is measuring the cytotoxic activity of the test agent against a cell comprised in the treatment resistant organoid.

Description

[Technical Field] Provided are methods for culturing a a tissue analogous to a cancer tissue, cell clusters obtained by the culturing methods, and their utilization. In particular, the present invention relates to a culture method for forming a treatment resistant organoid, wherein the method comprises a step of three-dimensionally culturing an organoid which was treated with at least one anticancer agent and isolating a treatment resistant organoid that is resistant to the treatment with said anticancer agent, wherein the step of three-dimensionally culturing is culturing in the absence of said at least one anticancer agent and wherein the organoid is formed by a pre-culture step of culturing one or more cancer stem cells. [Background Art] The number of deaths due to cancer continues to increase worldwide. For example, cancer has become the leading cause of death of Japanese people in Japan as well. For some cancers, screenings for early cancer detection and the spread of vaccines for preventing viral infections, which are cancer risk factors, are showing improvements in treatment results and preventive effects. Furthermore, application of new technologies is being tried out one after another in cancer treatments using anticancer agents, surgical procedures, or irradiation with radiation or proton beams. Among these various cancer treatment techniques, the use of anticancer agents is still one of the major options. In the treatment of cancer with anticancer drugs (chemotherapy), acquisition of anticancer agent resistance by cancers is one of the major causes hindering treatment, and elucidation of the mechanism is awaited. In the treatment of colorectal cancer for example, treatment efficiency has been enhanced through improvements in new agents and surgical excision methods. As with other organs, the so-called neoadjuvant therapy or conversion therapy, where a tumor is shrunk by drug therapy in advance and then targeted for surgery, has become widely used even for cancers that are difficult to be excised. Advances in treatment technologies have also increased the number of cases of cancers that were previously not subjected to surgical excision, but are now being treated. However, there are still many cases of recurrence despite the success in treatments. One of the causes of recurrence is considered to be the acquisition of anticancer agent resistance by cancers. As for the reason for resistance, it is assumed that anticancer agent treatment induces a group of cells that have acquired resistance and that manage to survive. However, regarding the origin of cells that acquire resistance, several different theories such as cancer stem cells (CSC) and epithelial mesenchymal transition (EMT) have been advocated, but there is still a great deal of ongoing debate. In addition, there are no research reports that have captured the substance of resistance in actual clinical settings. Understanding this mechanism is important in order to regulate the acquisition of anticancer agent resistance by cancer cells. The present inventors have previously obtained a specific antibody against LGR5 (leucine-rich repeat-containing G-protein-coupled receptor 5), which is a CSC marker for colorectal cancer, demonstrated that CSC can be detected with high sensitivity in clinical tissues, and also established a CSC cell line (NPL 1,NPL 2 and PTL 3). Furthermore, an attempt to culture CSC by combining various growth factors is known (PTL 1). A method of culturing epithelial stem cells in an extracellular matrix also containing a growth factor has also been proposed (PTL 2). However, these known CSCs and their culture techniques are limited to three-dimensional culture of CSCs, and the mechanism of how cancers acquire resistance to anticancer agents was not elucidated. [Citation List] [Patent Literature] [PTL 1] WO2012/168930[PTL 2] US2012/0196312[PTL 3] WO2013/062083 [Non-Patent Literature] [NPL 1] Kobayashi, S., et al. Stem cells 30, 2631-2644 (2012).[NPL 2] Yamazaki, M., et al. Acta histochemica et cytochemica 48, 159-164 (2015) [Summary of Invention] [Technical Problem] An objective of the present disclosure is to provide a technique that enables the construction of a model of cancer tissue that has acquired anticancer agent resistance. Alternatively, an objective of the present disclosure is to use the model to elucidate the process of acquiring anticancer agent resistance in a cancer tissue, and to provide a new cancer treating technique that targets a target molecule in an anticancer agent-resistant cancer tissue or a method for discovering a cancer treating technique that overcomes the resistance of a cancer to an anticancer agent. [Solution to Problem] The present inventors discovered that small cell clusters are formed in cultures when a cancer tissue or a tissue analogous to a cancer tissue which was treated with an anticancer agent is three-dimensionally cultured, and elucidated the properties thereof. The newly discover