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Article Open Access

Evaluation of HMGB1 as possible marker via breast organoid cultures research

  • Authors:
    • Vincenza Ciaramella
    • Barbara Carrese
    • Luigi Coppola
    • Giovanni Smaldone
    • Massimiliano D'Aiuto
    • Gennaro Mossetti
    • Andrea Soricelli
    • Marco Salvatore
  • View Affiliations / Copyright

    Affiliations: IRCCS SYNLAB SDN, I-80146 Naples, Italy, Clinica Villa Fiorita, I-81031 Aversa, Italy, Pathological Anatomy Service, Maria Rosaria Clinic, Pompei, I-80045 Naples, Italy
    Copyright: © Ciaramella et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 106
    |
    Published online on: July 1, 2025
       https://doi.org/10.3892/or.2025.8939
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Abstract

High mobility group box 1 (HMGB1) is a non‑histone protein widely expressed in the nucleus of mammalian cells, and it can be released by both immune and tumor cells. In the extracellular context HMGB1 can act as a proinflammatory mediator and boosting cancer progression. High HMGB1 mRNA expression levels are usually observed in various malignant diseases, including breast cancer (BC). Several studies have demonstrated the potential clinical value of HMGB1 in BC diagnosis and therapy. The present data, using in vitro protocols and molecular technologies, demonstrated the presence of HMGB1 in organoids derived from patients with BC with significantly elevated expression correlating with poorer prognosis. By blocking the activity of HMGB1 protein, cell cycle arrest and induction of apoptosis was observed in ex vivo 3D organoids, suggesting a potential antitumor effect. The localization by immunofluorescence, of HMGB1, β‑catenin and NF‑kB in organoids and the subsequent inhibition of the entire molecular pathway by switching off HMGB1 signaling suggests that there is a crosstalk between these molecules demonstrating their involvement in inflammation and inflammation‑associated diseases such as cancer. The current results aim to investigate the role of HMGB1 in BC progression and find innovative applications based on HMGB1 as a therapeutic target and early disease biomarker.
View Figures

Figure 1

Expression analysis of the HMGB gene
comparing values between healthy and tumour patients, 291 and 1,085
respectively. Data was reported on Gene Expression Profiling
Interactive Analysis (http://gepia2.cancer-pku.cn/#analysis). HMGB, High
mobility group box; BRCA, breast cancer; TPM, transcripts per
million.

Figure 2

RT-qPCR and immunofluorescence. (A)
RT-qPCR analysis of HMGB1 in two groups of PDOs: The first one
derived from fibroadenoma samples considered as non-cancerous
tissue sample (NC01-NC07) and the second one derived from tumor
tissues (1–7). Results were normalized to β-actin mRNA
and analyzed by the 2−ΔΔCq method. One-way ANOVA test
followed by Tukey's test were used for statistical analysis.
****P<0.001. (B) Representative images of HMGB1 localization in
tumor and non-cancerous ex vivo patient-derived organoids.
Scale bar, 10 µm. RT-qPCR, reverse transcription-quantitative PCR;
HMGB1, High mobility group box 1; NC, negative control.

Figure 3

Flow cytometry and RT-qPCR. (A) Flow
cytometric analysis of PDOs' apoptosis after treatment with
Glycyrrhizin for 72 h. Median values of apoptotic rate (%) is
reported in histogram plot (−=untreated control, +=Glycyrrhizin
treatment). ***P≤0.01. (B) Representative flow cytometry analysis
of the cell cycle distribution using Micheal H. Fox algoritm of
untreated (−)and Glycyrrhizin (+)-treated PDO. (C and D) RT-qPCR
analysis of Cyclin A, Cyclin B, Cyclin C and Cyclin D in (C)
non-cancerous and (D) tumor PDO samples treated with Glycyrrhizin
at 50 µM for 72 h. *P<0.05, ****P<0.0001. RT-qPCR, reverse
transcription-quantitative PCR; PDOs, patient-derived organoids;
CTRL, untreated control.

Figure 4

Expression analysis of of NF-κB and
β-catenin in tumor PDOs after 72-h treatment. Results were
normalized to β-actin mRNA and analyzed by the 2−ΔΔCq
method. One-way ANOVA test followed by Tukey's test were used for
statistical analysis. ***P<0.001. PDOs, patient-derived
organoids; CTRL, untreated control.

Figure 5

Representative images of our target
molecules through immunofluorescence analysis. (A-C) Localization
of (A) β-Catenin, (B) NF-κB and (C) α-HMGB1 in tumor ex vivo
PDOs before (CTRL) and after treatment with 50 µM of Glycyrrhizin,
direct HMGB1 inhibitor. Maximal projection images of PDO incubated
with β-Catenin (green signal), NF-κB (yellow signal) and α-HMGB1
(red spots). Cell nuclei were stained with Hoechst 33342 (blue
signal). Scale bar, 10 µm. The disappearance of (A) β-Catenin and
(B) NF-κB signals after treatment is shown; this effect is driven
by HMGB1, which is completely inactivated by (C) Glycyrrhizin. HMGB
1, High mobility group box 1; CTRL, untreated control.
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Copy and paste a formatted citation
Spandidos Publications style
Ciaramella V, Carrese B, Coppola L, Smaldone G, D'Aiuto M, Mossetti G, Soricelli A and Salvatore M: Evaluation of HMGB1 as possible marker via breast organoid cultures research. Oncol Rep 54: 106, 2025.
APA
Ciaramella, V., Carrese, B., Coppola, L., Smaldone, G., D'Aiuto, M., Mossetti, G. ... Salvatore, M. (2025). Evaluation of HMGB1 as possible marker via breast organoid cultures research. Oncology Reports, 54, 106. https://doi.org/10.3892/or.2025.8939
MLA
Ciaramella, V., Carrese, B., Coppola, L., Smaldone, G., D'Aiuto, M., Mossetti, G., Soricelli, A., Salvatore, M."Evaluation of HMGB1 as possible marker via breast organoid cultures research". Oncology Reports 54.3 (2025): 106.
Chicago
Ciaramella, V., Carrese, B., Coppola, L., Smaldone, G., D'Aiuto, M., Mossetti, G., Soricelli, A., Salvatore, M."Evaluation of HMGB1 as possible marker via breast organoid cultures research". Oncology Reports 54, no. 3 (2025): 106. https://doi.org/10.3892/or.2025.8939
Copy and paste a formatted citation
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Spandidos Publications style
Ciaramella V, Carrese B, Coppola L, Smaldone G, D'Aiuto M, Mossetti G, Soricelli A and Salvatore M: Evaluation of HMGB1 as possible marker via breast organoid cultures research. Oncol Rep 54: 106, 2025.
APA
Ciaramella, V., Carrese, B., Coppola, L., Smaldone, G., D'Aiuto, M., Mossetti, G. ... Salvatore, M. (2025). Evaluation of HMGB1 as possible marker via breast organoid cultures research. Oncology Reports, 54, 106. https://doi.org/10.3892/or.2025.8939
MLA
Ciaramella, V., Carrese, B., Coppola, L., Smaldone, G., D'Aiuto, M., Mossetti, G., Soricelli, A., Salvatore, M."Evaluation of HMGB1 as possible marker via breast organoid cultures research". Oncology Reports 54.3 (2025): 106.
Chicago
Ciaramella, V., Carrese, B., Coppola, L., Smaldone, G., D'Aiuto, M., Mossetti, G., Soricelli, A., Salvatore, M."Evaluation of HMGB1 as possible marker via breast organoid cultures research". Oncology Reports 54, no. 3 (2025): 106. https://doi.org/10.3892/or.2025.8939
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