Hedgehog Signals Mediate Anti-Cancer Drug Resistance in Three-Dimensional Primary Colorectal Cancer Organoid Culture

Abstract

Colorectal cancer is one of the most common causes of cancer death worldwide. In patients with metastatic colorectal cancer, combination treatment with several anti-cancer drugs is employed and improves overall survival in some patients. Nevertheless, most patients with metastatic disease are not cured owing to the drug resistance. Cancer stem cells are known to regulate resistance to chemotherapy. In the previous study, we established a novel three-dimensional organoid culture model from tumor colorectal tissues of human patients using an air–liquid interface (ALI) method, which contained numerous cancer stem cells and showed resistance to 5-fluorouracil (5-FU) and Irinotecan. Here, we investigate which inhibitor for stem cell-related signal improves the sensitivity for anti-cancer drug treatment in tumor ALI organoids. Treatment with Hedgehog signal inhibitors (AY9944, GANT61) decreases the cell viability of organoids compared with Notch (YO-01027, DAPT) and Wnt (WAV939, Wnt-C59) signal inhibitors. Combination treatment of AY9944 or GANT61 with 5-FU, Irinotecan or Oxaliplatin decreases the cell viability of tumor organoids compared with each anti-cancer drug alone treatment. Treatment with AY9944 or GANT61 inhibits expression of stem cell markers c-Myc, CD44 and Nanog, likely through the decrease of their transcription factor, GLI-1 expression. Combination treatment of AY9944 or GANT61 with 5-FU or Irinotecan also prevents colony formation of colorectal cancer cell lines HCT116 and SW480. These findings suggest that Hedgehog signals mediate anti-cancer drug resistance in colorectal tumor patient-derived ALI organoids and that the inhibitors are useful as a combinational therapeutic strategy against colorectal cancer.

Establishment of a novel experimental model for muscle‐invasive bladder cancer using a dog bladder cancer organoid culture

“…To demonstrate the use of BC organoids as a preclinical model for checking the sensitivity of anticancer drugs, we performed a 96‐well Matrigel cell viability assay, as described previously . Treatment with a nonselective cyclooxygenase (COX) inhibitor, piroxicam, had no effect on the cell viability of each BC organoid (Figure A,B).…”

“…Cell viability assay of organoids was performed as described previously . Taking into consideration the therapeutic dose in clinic and pharmacokinetics in dogs, we determined the appropriate concentration to be used for each drug in the cell viability assay.…”

Patient-derived organoid analysis of drug resistance in precision medicine: is there a value?

“…In colorectal cancer (CRC), PDO culture has been used to analyzes the mechanism of drug resistance. Using an air-liquid interface method, Usui et al [21,33], developed a new CRC organoid culture contained a lot of CSCs and showed resistance to 5-fluorouracil, and irinotecan by activation of hedgehog signaling of CSCs. Moreover, treatment with hedgehog signal inhibitors (AY9944, GANT61) diminished the cell viability of these organoids [21].…”

“…Cancer stem cells (CSCs) are tumor-initiating cells capable of conserving cellular heterogeneity, self-renewal, epithelial to mesenchymal transition, differentiation to form all kind cells in a given tumor [12] and drive the tumor growth, metastasis and most importantly, the resistance to conventional anticancer agents [13][14][15][16][17][18][19][20][21][22]. Therefore, the total eradication of CSCs is crucial for the successful treatment of cancers.…”

“…Among them, organoids constitute the more reasonable method in recapitulating the in vivo microenvironment of the tumor, and easier handling as well as high throughput screening than slice cultures or xenografts. During the past decade, researchers have made substantial progress in the field of organoid biology, and organoid systems have already been reviewed in detail elsewhere [21,23,24].…”