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Short summary of the research and contribution to the field

This preprint investigates the mechanism by which a recombinant measles virus armed with the pro-apoptotic gene BNiP3 / BNIP3 may exert anti-tumor effects in triple-negative breast cancer cells. The authors use bioinformatics analysis of TNBC versus non-TNBC cell-line expression data to identify candidate regulatory genes, highlighting CTNNB1, which encodes β-catenin, as a key hub gene. They then evaluate whether infection with recombinant measles virus carrying BNiP3 reduces β-catenin signaling and related downstream targets in breast cancer cell lines. The study reports that rMV-BNiP3 infection reduces β-catenin expression, downregulates downstream targets such as cyclin D1, MMP7, and c-JUN, reduces ERK/AKT pathway activation, and suppresses migration/spheroid growth more prominently in MDA-MB-231 TNBC cells than in MCF-7 cells.

The work is relevant because TNBC remains an aggressive and therapy-resistant breast cancer subtype with limited targeted-treatment options. The manuscript contributes to the field by connecting an engineered oncolytic measles-virus approach with a plausible TNBC-relevant pathway, Wnt/β-catenin signaling, and by attempting to provide mechanistic support for prior observations that rMV-BNiP3 has selective anti-proliferative activity in TNBC cells.

Positive feedback / strengths

1. Clinically relevant therapeutic concept. Oncolytic virotherapy is an important emerging area, and applying an engineered measles-virus platform to TNBC is a meaningful direction given the aggressive biology and therapeutic limitations of TNBC.

2. Mechanistic focus beyond cytotoxicity. The manuscript does not stop at showing cell death or reduced proliferation; it attempts to connect rMV-BNiP3 activity to a known cancer-relevant pathway, β-catenin/Wnt signaling, and downstream mediators of proliferation and invasion.

3. Use of both bioinformatics and experimental validation. The authors combine network-based hub-gene identification with qRT-PCR, western blotting, wound-healing assays, and 3D spheroid culture. This multi-layered approach strengthens the overall study design.

4. Relevant cell-line model comparison. The comparison between MDA-MB-231 as a TNBC/invasive model and MCF-7 as a non-TNBC/luminal model is a reasonable first-pass experimental system.

5. Useful visual evidence. Figure 1 on page 16 shows CTNNB1, EGFR, CDH1, and JUN as hub genes with qRT-PCR validation; Figure 3 on page 18 shows reduced β-catenin after rMV-BNiP3 infection in MDA-MB-231 cells; Figure 4 on page 19 shows reduced β-catenin downstream targets; and Figure 7 on page 22 shows spheroid-size changes after infection. These figures help readers follow the proposed mechanism.

Major issues

1. The central mechanistic claim needs stronger causal evidence

The manuscript shows that rMV-BNiP3 infection is associated with reduced β-catenin and downstream targets, but it does not yet prove that β-catenin pathway suppression is the direct mechanism driving reduced migration, proliferation, or cell death.

Suggested improvement: To strengthen causality, the authors should consider:

• β-catenin rescue or overexpression after rMV-BNiP3 infection

• CTNNB1 knockdown as a positive control for pathway suppression

• Wnt/β-catenin pathway activation, such as Wnt ligand or GSK3β inhibition, to test whether pathway reactivation rescues phenotype

• TOPFlash/FOPFlash β-catenin transcriptional reporter assay

• nuclear versus cytoplasmic β-catenin localization by immunofluorescence

• assessment of active/non-phosphorylated β-catenin

Without these experiments, the current data support an association between rMV-BNiP3 infection and β-catenin reduction, but not a definitive mechanism.

2. Cytotoxicity and migration effects may be confounded

The wound-healing and spheroid results suggest reduced migration and growth after rMV-BNiP3 infection. However, because the virus is cytotoxic/pro-apoptotic, reduced wound closure or spheroid expansion may reflect reduced viability or proliferation rather than a specific anti-migratory effect.

Suggested improvement: The authors should include:

• cell viability measurements at the same timepoints as migration assays

• proliferation-normalized migration analysis

• transwell migration/invasion assays

• Matrigel invasion assay

• wound-healing quantification using percent wound closure

• mitomycin C or other proliferation-control strategy, if appropriate

• apoptosis markers such as cleaved caspase-3, cleaved PARP, Annexin V/PI, or TUNEL

This would help distinguish true migration/invasion suppression from general cytotoxicity.

3. Viral infection parameters and controls need more detail

The manuscript describes infection with purified rMV and rMV-BNiP3, but the infection conditions are not fully detailed. For an oncolytic-virus study, reproducibility depends heavily on viral dose, titer, multiplicity of infection, infection duration, and viral replication kinetics.

Suggested improvement: The authors should report:

• viral titer and unit used

• multiplicity of infection

• infection duration for each assay

• whether equal infectious units of rMV and rMV-BNiP3 were used

• viral replication kinetics in MDA-MB-231 versus MCF-7

• number of biological replicates

• whether UV-inactivated virus or heat-inactivated virus was tested

• whether BNiP3 expression level correlates with cytotoxicity/pathway suppression

This would make the viral biology and comparison between rMV and rMV-BNiP3 more interpretable.

4. Selectivity for TNBC requires broader validation

The study uses one TNBC cell line, MDA-MB-231, and one non-TNBC cell line, MCF-7. While this is a useful starting point, TNBC is highly heterogeneous, and conclusions about TNBC selectivity cannot be made from one TNBC model alone.

Suggested improvement: The authors should validate the findings in additional cell lines, such as:

• TNBC: MDA-MB-468, BT-549, HCC1806, HCC1937, Hs578T

• non-TNBC/luminal/HER2-positive: T47D, ZR-75-1, BT-474, SKBR3

• non-malignant mammary epithelial cells, such as MCF10A, as a safety/selectivity control

This would help determine whether the effect is truly TNBC-biased or specific to MDA-MB-231 cells.

5. CD46 receptor analysis is insufficient to explain infection bias

The authors evaluate CD46 expression and note only a slight, non-significant increase in MDA-MB-231 cells. This is helpful, but it does not fully explain why rMV-BNiP3 appears more effective in TNBC cells.

Suggested improvement: The authors should expand this section by testing:

• CD46 surface expression by flow cytometry

• viral entry efficiency

• viral genome copy number over time

• viral protein expression kinetics

• receptor-blocking experiments

• other possible determinants of measles-virus sensitivity, such as innate antiviral response, interferon signaling, apoptosis threshold, or metabolic state

The current CD46 western blot alone is not enough to explain cell-type selectivity.

6. Western blot and qPCR data need stronger quantification and statistical reporting

The manuscript includes important western blots and qRT-PCR data, but many results appear qualitative or semi-quantitative. Several graphs show densitometry, but it is unclear how many biological replicates were performed or whether statistical testing was applied.

Suggested improvement: For all qRT-PCR and western blot data, the authors should report:

• number of biological replicates

• number of technical replicates

• error bars

• statistical tests

• p-values

• normalization method

• full-length uncropped blots in supplement

• loading-control consistency

• whether densitometry was performed across multiple independent experiments

This is particularly important for the β-catenin, cyclin D1, MMP7, c-JUN, pERK, and pAKT claims.

7. Bioinformatics analysis is under-described and may be too narrow

The bioinformatics analysis appears to compare MDA-MB-231 and MCF-7 cell-line data and identify hub genes using PPI topology. This is a narrow comparison and may reflect differences between two cell lines rather than general TNBC biology.

Suggested improvement: The authors should clarify:

• dataset accession or database source

• expression data type and preprocessing

• DEG thresholds

• number of DEGs used in STRING

• STRING confidence cutoff

• whether the network used experimental/validated interactions only or all predicted interactions

• whether patient tumor datasets were used for validation

• whether CTNNB1 is elevated in TNBC patient samples, not only MDA-MB-231 cells

Including TCGA/METABRIC/CPTAC or other TNBC patient datasets would strengthen the relevance of CTNNB1 as a TNBC-related target.

8. BNiP3-specific contribution needs clearer separation from measles-virus effect

The manuscript compares rMV and rMV-BNiP3 in several experiments, which is good. However, it remains unclear whether the observed β-catenin pathway suppression is due specifically to BNiP3 expression, general measles-virus infection, differential viral replication, or increased cytotoxic stress.

Suggested improvement: Useful controls would include:

• rMV without BNiP3 at matched viral replication level

• BNiP3 expression alone without virus

• catalytically/functional-domain altered BNiP3 construct, if applicable

• time-course analysis of BNiP3 expression versus β-catenin reduction

• apoptosis rescue or caspase-inhibition experiments

• viral replication-normalized pathway analysis

This would help define whether BNiP3 is the driver or enhancer of the observed pathway effects.

9. In vivo and normal-cell safety data are needed before therapeutic claims

The manuscript suggests that rMV-BNiP3 could be an effective treatment modality for invasive TNBC. This is promising, but the current data are mostly in vitro and based on limited cell-line models.

Suggested improvement: The authors should soften therapeutic claims and state that the data support preclinical in vitro potential. Future validation should include:

• xenograft or orthotopic TNBC models

• tumor biodistribution

• viral replication in tumor versus normal tissue

• immune response considerations

• toxicity assessment

• normal mammary epithelial-cell controls

• dose-response and route-of-administration studies

• comparison with standard therapies or combination therapy

The manuscript mentions that in vivo validation is ongoing, which is encouraging, but current conclusions should remain cautious.

Minor issues

1. Standardize BNiP3 / BNIP3 spelling. The manuscript uses BNiP3, BNIP3, and sometimes rMV-BNIP3. Please use one consistent gene/protein nomenclature, ideally BNIP3 for the gene/protein and rMV-BNIP3 or rMV-BNiP3 consistently for the virus.

2. Correct typographical errors. Examples include “Emal” instead of “Email,” “rMB-BNiP3” instead of rMV-BNiP3, and other formatting inconsistencies.

3. Clarify MCF-7 labeling. Some figure text refers to “MCF-7 non-invasive TNBC cell line,” which is incorrect. MCF-7 should be described as a non-TNBC/luminal breast cancer cell line.

4. Clarify timepoints. Some sections mention 24 hours, while others mention 48 hours post-infection. Please clearly state which assays were performed at which timepoint.

5. Improve figure resolution and labeling. Some blot labels and densitometry axes are difficult to read. Higher-resolution figures and clearer labeling would help.

6. Add scale bars to microscopy images. Wound-healing and spheroid images should include scale bars and quantification.

7. Quantify spheroid size. Figure 7 is visually useful, but spheroid diameter, area, circularity, and invasion/migration area should be quantified across biological replicates.

8. Clarify whether 3D spheroid assay measures migration, growth, or viability. The spheroid assay is described as migration/proliferation-related. A clearer endpoint definition would help.

9. Improve reference accuracy. Some references appear to have incorrect years or formatting. For example, the classic Foulkes NEJM TNBC review is not from 2026. The reference list should be checked carefully.

10. Add data availability statement. The authors should provide access to raw qPCR data, densitometry values, image quantification, and bioinformatics workflow/code where possible.

Overall assessment

This is an interesting and potentially valuable preclinical study linking an engineered oncolytic measles virus carrying BNIP3 to suppression of β-catenin-related signaling in TNBC cells. The manuscript’s strengths include its focus on a clinically challenging breast cancer subtype, use of a mechanistic pathway hypothesis, comparison of rMV and rMV-BNiP3, and inclusion of both 2D and 3D experimental models.

The major improvements needed are stronger causal evidence that β-catenin suppression drives the observed anti-tumor phenotype, clearer viral-infection parameters, broader validation across multiple TNBC and non-TNBC cell lines, better quantification/statistics, and stronger separation of BNiP3-specific effects from general measles-virus cytotoxicity. The current data support rMV-BNiP3 as a promising in vitro preclinical strategy, but therapeutic claims should remain cautious until expanded cell-line, normal-cell, mechanistic, and in vivo validation is available.

With these revisions, the manuscript would provide a stronger mechanistic foundation for developing rMV-BNiP3 as a potential oncolytic virotherapy platform for TNBC.

Competing interests

The author declares that they have no competing interests.

Use of Artificial Intelligence (AI)

The author declares that they used generative AI to come up with new ideas for their review.