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.
Introduction
Itraconazole (ITZ) is a long-established and widely prescribed antifungal agent that inhibits ergosterol synthesis in fungal cell membranes. In addition to its antifungal effects, ITZ has demonstrated antitumor activity in various solid malignancies, including basal cell carcinoma, prostate cancer, non-small cell lung cancer, and pancreatic cancer (1). The mechanisms underlying its antitumor effects are multifaceted and include inhibition of intracellular signaling pathways such as Akt/mammalian target of rapamycin (mTOR), Sonic Hedgehog, and Wnt/β-catenin; suppression of mitochondrial voltage-dependent anion channel 1; and interference with lipid transport proteins, including sterol carrier protein-2 and Niemann-Pick disease type C1.
Tumor-associated macrophages (TAMs) are key components of the tumor microenvironment and predominantly exhibit an M2 phenotype, which promotes tumor progression by supporting angiogenesis, immunosuppression, and metastasis. In contrast, M1 macrophages exert anti-tumor effects by producing pro-inflammatory cytokines and enhancing cytotoxic T cell responses. Although several clinical trials have aimed to reprogram TAMs from the M2 to the M1 phenotype, most have shown only modest efficacy or have been associated with significant toxicity, underscoring the need for more effective and safer therapeutic strategies (2).
Previously, we demonstrated that itraconazole (ITZ) reprograms TAMs from the M2 to the M1 phenotype using THP-1-derived macrophages, a widely accepted in vitro model of TAMs in the tumor microenvironment. ITZ-induced M1-like macrophages exhibited suppressive effects on cancer cell proliferation. This phenotypic repolarization was reversible and sustained with continued ITZ exposure (3, 4). Mechanistically, the repolarization was mediated by ITZ-induced modulation of lysosomal cholesterol release (5).
In the present study, we investigated whether ITZ induces M1 polarization of TAMs in human tissues. We conducted a window-of-opportunity (WOO) trial to evaluate the clinical effects and elucidate the mechanisms of action of ITZ in patients with solid tumors. Paired tumor biopsy specimens obtained before and after ITZ treatment were subjected to this study.
The WOO trial was registered in the University Hospital Medical Information Network (UMIN000018388) and the Japan Registry of Clinical Trials (jRCTs051190006). Written informed consent was obtained from all participants. This study was conducted as part of the WOO trial and its subsequent exploratory analyses, with approval from the institutional review board (approval number:5120).
Patients and Methods
Patient selection and treatment. Patients with advanced or recurrent cervical, vaginal, or vulvar cancer who were enrolled in the window-of-opportunity (WOO) trial were selected for this study, as changes in tumor size could be readily and objectively assessed within a short time frame using transvaginal ultrasonography or visual inspection. Itraconazole was administered as a 10 mg/ml oral solution at a dose of 20 ml twice daily for 1 to 4 weeks (Figure 1). Tumor response was evaluated using transvaginal ultrasonography during treatment. Patient symptoms and clinical background were retrospectively reviewed from medical records. Many patients experiencing genital bleeding and/or pain reported symptom relief within two weeks of initiating oral itraconazole therapy. Although these observations were anecdotal and subjective, they were included in the analysis due to their perceived clinical relevance. Tumor size and symptomatic response to ITZ were assessed after one or two weeks of treatment. Adverse events during ITZ treatment were also assessed according to the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0.
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.
Immunohistochemical analysis of tumor-associated macrophages. Paired tumor biopsy specimens obtained before and after ITZ treatment from patients who exhibited a ≥20% reduction in tumor diameter within two weeks were subjected to immunohistochemical analysis. Formalin-fixed, paraffin-embedded tumor sections were cut at a thickness of 4 μm. Sections were incubated with a rabbit monoclonal anti-CD163 antibody (D6U1J, Cell Signaling Technology, Danvers, MA, USA) and a mouse monoclonal anti-CD86 antibody (C86/1146, Abcam, Cambridge, UK). Chromogenic detection was performed using ImmPACT DAB EqV Substrate (Vector Laboratories, Newark, CA, USA) until optimal staining intensity was achieved, followed by ImmPACT Vector Red Substrate for dual labeling. Nuclear counterstaining was carried out with hematoxylin (FUJIFILM Wako, Osaka, Japan).
Stained slides were analyzed using an automated quantitative pathology imaging system (Vectra3, AKOYA Biosciences, Marlborough, MA, USA). For each paired specimen from the three responsive patients, five randomly selected high-power fields (20× Objective) were analyzed per sample, focusing on immune cell infiltrates associated with tumor nests. These regions included both tumor parenchyma and adjacent stromal areas. Nuclei were automatically segmented, and staining intensities were quantified radially from the nuclear center. Cells positive for FAST RED (CD163) and DAB (CD86) were identified, and signal intensities were quantified using inForm software version 2.6 (AKOYA Biosciences).
Statistical analysis. Differences in the proportions of cell subtypes between pre-treatment and post-treatment groups were evaluated using the Mann-Whitney U test with Prism 9 software (GraphPad Software, La Jolla, CA, USA). For each case, 4-6 high-power fields (HPFs) were analyzed at each time point, and HPFs were treated as independent samples. A p-value of <0.05 was considered statistically significant.
Results
Between September 2015 and November 2023, 19 patients with cervical, vaginal and vulvar cancer were enrolled in the WOO trial. Baseline patient characteristics are summarized in Table I. Four patients [21.1%; 95% confidence interval (CI)=6.1-45.6%] exhibited a rapid response to ITZ defined as a ≥20% reduction in tumor diameter within two weeks. In one patient with cervical adenocarcinoma, the tumor showed complete macroscopic regression after two weeks of ITZ treatment, precluding post-treatment tissue sampling (Table II). For the remaining three patients, paired tumor biopsy specimens obtained before and after itraconazole treatment were subjected to immunohistochemical analysis. Fourteen out of 17 symptomatic patients (82.4%; 95%CI=56.6%-96.2%) reported symptom improvement within two weeks of ITZ administration. Adverse events occurred in two patients. One patient experienced grade 1 malaise, and the other developed grade 3 diarrhea, defined as more than 10 episodes per day. Both patients discontinued ITZ treatment, with the latter doing so on day 3 of administration.
Patient characteristics.
Response during itraconazole (ITZ) treatment.
Among the three patients with paired tumor biopsies, quantitative immunohistochemical analysis demonstrated a significant increase in CD86 single-positive (M1-like) TAMs after itraconazole treatment when evaluated using the Mann-Whitney U test treating HPFs as independent samples (Figure 2). In a representative responder shown in Figure 3A and B–a 75-year-old woman with stage IIIB cervical squamous cell carcinoma–vaginal bleeding decreased, and the maximum tumor diameter, as measured using vaginal ultrasound, was reduced from 61 mm to 49 mm after one week of itraconazole treatment (Figure 3, Figure 4).
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.
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).
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.
Discussion
To the best of our knowledge, this is the first report to demonstrate that oral itraconazole administration induces a shift from M2- to M1-like TAMs in human cancer tissues. Interestingly, the population of CD163 single-positive M2-like macrophages did not decrease following treatment, suggesting that the increase in M1-like macrophages may result not only from repolarization of resident M2 macrophages but also from the recruitment of peripheral M1 macrophages.
In the representative responder shown in Figure 3A and B, transcriptomic analysis of paired tumor samples revealed that matrix metalloproteinases 1 (MMP1) expression was reduced by more than 256-fold, while MMP3, MMP10, and MMP13 were each downregulated by approximately 64-fold. These changes are consistent with M1 effects (data not shown).
Itraconazole has been reported to exhibit potent anti-angiogenic activity (6), which may contribute to both symptom relief–such as reduced genital bleeding–and tumor shrinkage. Furthermore, in cervical cancer cell lines such as CaSki cells harboring mutations in PIK3CA at codon E545K and Sonic Hedgehog at codon D154E, itraconazole has been shown to suppress the Akt/mammalian target of rapamycin (mTOR) and Sonic Hedgehog signaling pathways, thereby inhibiting tumor cell proliferation (7). The tumor shown in Figure 3A and B had pathogenic variants in PIK3CA (E542K, E545K) and PTEN (E242*, Q245*) revealed in daily practice genomic profiling.
In the present study, two out of three patients with melanoma exhibited tumor decrease and reduced genital bleeding. One patient with vaginal melanoma showed decreased accumulation of fluorine-18fluorodeoxyglucose in positron emission tomography/computed tomography imaging, as previously reported (8). In established melanoma cell lines, ITZ directly suppressed tumor growth by inhibiting the Hedgehog signaling pathway (9). These findings suggest that itraconazole may exert both immunomodulatory and direct antitumor effects in patients with cancer.
One limitation of our study is that immunohistochemical analysis was not performed on tumor samples from non-responders, precluding a comparative evaluation of macrophage polarization between responders and non-responders. In addition, CD86 was selected as a representative M1 marker; however, while it is widely used, CD86 is not specific to macrophages and can also be expressed by other immune cells, such as dendritic cells. Ideally, true M1 macrophages should be identified as CD68 and CD86 double-positive cells. Future studies employing more specific multi-marker strategies–such as multiplex immunofluorescence or single-cell RNA sequencing–are warranted to refine the classification of TAM subsets.
The strengths of this study include the rapid tumor responses and symptom relief observed after short-term itraconazole treatment, along with clear evidence of M1 polarization in tumor tissues from responders. However, the WOO trial was not designed for assessment of long-term efficacy, and the small sample size limits generalizability. Further investigation in a larger cohort is warranted.
In conclusion, short-term oral itraconazole treatment was associated with increased infiltration of M1-like TAMs in cervical cancer tissues, supporting its potential as an immunomodulatory anticancer agent.
Footnotes
Authors’ Contributions
HT and TU conceived and designed the study. All Authors contributed to data analysis and interpretation and approved the final version of the manuscript.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
Funding
This work was supported by “Hyogo Medical University Diversity Grant for Research Promotion” under MEXT Funds for the Development of Human Resources in Science and Technology, “Initiative for Realizing Diversity in the Research Environment (Characteristic-Compatible Type)”.
Artificial Intelligence (AI) Disclosure
No sections involving the generation, analysis, or interpretation of research data were produced by generative AI. All scientific content was created and verified by the authors. Furthermore, no figures or visual data were generated or modified using generative AI or machine learning-based image enhancement tools.
- Received August 26, 2025.
- Revision received November 14, 2025.
- Accepted November 17, 2025.
- Copyright © 2026 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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