Oral Administration of Itraconazole Induces M1 Polarization of Tumor-associated Macrophages in Gynecological Cancer

Abstract

Background/aim: Itraconazole (ITZ), an antifungal agent with reported anticancer properties, has been shown to induce phenotypic repolarization of tumor-associated macrophages (TAMs) from an M2-like to an M1-like phenotype in vitro. This study aimed to evaluate the clinical response to oral ITZ and its effects on TAM polarization in human tumor tissues.

Patients and methods: Nineteen patients with cervical, vaginal, or vulvar cancer received oral ITZ (20 ml of 10 mg/ml solution, twice daily) in a window-of-opportunity trial (jRCTs051190006). Tumor response was assessed using transvaginal ultrasound. Paired tumor biopsy specimens obtained before and after ITZ treatment from patients with ≥20% tumor reduction within two weeks of ITZ treatment were analyzed by immunohistochemistry using anti-CD163 and anti-CD86 antibodies to identify M2-like and M1-like TAMs, respectively. Quantitative image analysis was performed using the Vectra3 system and inForm software.

Results: Among the 19 patients, four [21.1%; 95% confidence interval (CI)=6.1-45.6%] showed ≥20% tumor reduction within two weeks of ITZ treatment, including one patient who achieved complete macroscopic regression. Immunohistochemical analysis of paired tumor samples from the remaining three responders demonstrated an increase in CD86 single-positive and CD163/CD86 double-positive TAMs after ITZ administration.

Conclusion: ITZ treatment was associated with increased infiltration of M1-like TAMs in cancer tissues, suggesting an immunomodulatory effect. These findings support further investigation of ITZ as a potential adjunct in cancer therapy.

Keywords: Itraconazole; M1 polarization; cervical cancer; repurposing; tumor-associated macrophage.

Figure 1

Schema of a window-of-opportunity trial for repurposing itraconazole as an anticancer drug. This trial was prospectively registered with the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR; registration number UMIN000018388) in August 2015, and with the Japan Registry of Clinical Trials (jRCT; registration number jRCT051190006) in April 2019. Post-treatment tumor biopsies could be substituted with surgical specimens. The collected samples were subjected to multi-omics analyses to identify biomarkers and therapeutic targets of itraconazole. Following a protocol amendment in July 2023, post-treatment biopsies were no longer required.

Figure 2

Quantitative comparison of macrophage subpopulations before and after itraconazole treatment in rapid responders (n=3). Box plots illustrate the proportions of macrophage phenotypes –CD163⁺/CD86⁻, CD163⁺/CD86⁺, and CD163⁻/CD86⁺– in tumor tissues before (Pre, light gray) and after (Post, dark gray) itraconazole treatment. Between-group differences were tested using the Mann-Whitney U test treating high-power fields (HPFs) as independent samples (p-values shown). CD163⁺/CD86⁺ and CD163⁻/CD86⁺ increased significantly after itraconazole treatment. Significant increases were observed in the CD163⁺/CD86⁺ population (p<0.001) and the CD163⁻/CD86⁺ population (p=0.017) after itraconazole treatment.

Figure 3

Representative histological findings of M1 repolarization. (A, B) Bright-field images of tumor tissue obtained before (A) and after (B) itraconazole treatment. CD163-positive cells were stained with Fast Red, CD86-positive cells with DAB, and nuclei were counterstained with hematoxylin. (C, D) Color-decomposed images corresponding to panels A and B, respectively. Cells were classified and color-coded based on CD163 and CD86 expression as follows: CD163⁺/CD86⁻ (red), CD163⁺/CD86⁺ (yellow), CD163⁻/CD86⁺ (green), and CD163⁻/CD86⁻ (blue).

Figure 4

Quantitative analysis of tumor-associated macrophage populations in a representative case. Box plots illustrate the relative proportions of macrophage subpopulations before and after itraconazole treatment, corresponding to the histological images in Figure 3. The percentage shown in the box represents the average calculated from five randomly selected high-power fields (20× Objective) including both tumor parenchyma and adjacent stromal areas.