# Resolving tumor microenvironment heterogeneity to forecast immunotherapy response in triple-negative breast cancer through multi-scale analysis

Researcher: Ge Zhang
Chinese name: 张格
ORCID identity anchor: https://orcid.org/0000-0002-3116-3246
Canonical researcher: https://drgezhang.com/#person

Journal: Frontiers in Oncology
Year: 2025
Type: Article
DOI: 10.3389/fonc.2025.1538574
Canonical page: https://drgezhang.com/papers/doi-10-3389-fonc-2025-1538574.html
Markdown record: https://drgezhang.com/papers/doi-10-3389-fonc-2025-1538574.md

## Full Authors

1. Shihao Sun
2. Shuang Chen
3. Kaiyuan Li
4. Ge Zhang
5. Nan Wang
6. Yijia Xu
7. Xinxing Wang
8. Jiangrui Chi
9. Lin Li
10. Yi Sun

<a id="paper-discovery"></a>
## Research & review context

### Research fields

- Breast cancer single-cell genomics
- Immunotherapy response modeling

### Review topics

- BRCA1-associated TNBC microenvironment
- ISG15 and cancer-associated fibroblasts

### Scientific role

Exploratory scRNA/spatial/proteomic TNBC comparison with mixed-cancer ICI-response modeling.

### Evidence position

Small human donor-level omics associations and modest cross-cancer classifier; no TNBC-treated external validation.

### Related concepts

- BRCA1-WT myCAF states
- ISG15
- No TNBC ICI-treated test

### Possible citation contexts

- Reviews describing CAF and T-cell state hypotheses in BRCA1-stratified TNBC single-cell evidence.
- Methodological discussions separating cross-cancer ICI-model AUC from genuine TNBC treatment-response validation.

Possible literature-review contexts, not evidence of existing citations. Read the findings and Evidence Scope before citing the original article.

## Evidence Scale

**Resolving tumor microenvironment heterogeneity to forecast immunotherapy response in triple-negative breast cancer through multi-scale analysis**

An integrative TNBC study compared four BRCA1-mutant and four BRCA1-wild-type single-cell specimens (32,386 cells), mapped CAF/immune-state differences, and nominated ISG15-associated myofibroblastic features using transcriptomic, spatial and proteomic analyses. Its immunotherapy response model was trained on mixed non-TNBC treatment cohorts and attained modest internal AUCs around 0.60–0.61; no treated TNBC validation cohort was established.

| Evidence | Value |
| --- | --- |
| TNBC single-cell donors | 8 specimens: 4 BRCA1-mutant and 4 BRCA1 wild-type |
| Cells after QC | 32,386; 18,182 BRCA1-mutant and 14,204 BRCA1 wild-type |
| Spatial specimens | 4 BRCA1-WT TNBC spatial samples; no matched BRCA1-MT spatial series in this analysis |
| Paired proteomics | 9 tumor samples and 9 corresponding para-tumor specimens; nine pairs, not 18 independent patients |
| ICI model pooled subset | Four external treatment cohorts pooled to 661 records and divided 80:20 internally |
| KNN classifier discrimination | Reported validation AUC 0.60 (95% CI 0.51–0.69); testing AUC 0.61 (95% CI 0.51–0.71) |

### Counting note

Cell profiles, donor tumor specimens, spatial samples, paired proteomic tissues and external cross-cancer treated cohorts are distinct counting units and may involve overlaps or subsets. The article inconsistently describes 13 ICI pre-treatment cohorts (829 records) in Methods versus 12 in Results; no unsupported sum or cohort count is promoted.

## Research Question

Which immune and stromal cell-state differences accompany BRCA1 status in sampled TNBC, how was ISG15 prioritized, and what evidence actually supports transferring an immunotherapy response model to TNBC?

## Author Evidence Summary

The investigators analyzed GSE161529 single-cell tumor specimens, mapped four BRCA1-wild-type spatial samples, integrated expression and an in-house paired proteomic cohort, and trained a KNN treatment-response classifier on several external ICI cohorts. The survival association in TCGA TNBC was not significant, and the response model was not validated on ICI-treated TNBC patients.

## Key Findings

<a id="kf1"></a>
### KF1: The TNBC single-cell series revealed BRCA1-associated differences in annotated tumor immune and stromal states.

Context: Observed four versus four specimens allow exploratory group comparison; 32,386 cells are nested within eight donors and are not independent patients.

| Evidence | Value |
| --- | --- |
| TNBC samples | 4 BRCA1-MT and 4 BRCA1-WT |
| QC-retained cells | 32,386; 18,182 mutant-group and 14,204 wild-type-group |
| Broad annotated lineages | 7 principal populations |

Source locator: PMC12401915: Methods Data acquisition and processing (s2_1); Results The single-cell atlas of BRCA1 MT and BRCA1 WT samples (s3_1); Figure 1A–D.

<a id="kf2"></a>
### KF2: T-cell exhaustion and dendritic/macrophage-associated expression programs differed by BRCA1 group.

Context: State labels and inferred scores are descriptive transcriptomic signatures; lineage transitions and immunotherapy effectiveness cannot be inferred directly.

| Evidence | Value |
| --- | --- |
| Comparison | BRCA1-WT versus BRCA1-MT single-cell specimens |
| CD4/CD8 states | Treg, Tex and other computationally annotated subsets |
| Intercellular readouts | Pseudotime and inferred spatial signaling, not direct lineage tracing |

Source locator: PMC12401915: Results T cells in BRCA1-WT specimens (s3_2); Activated TAMs and tolerogenic DCs (s3_3); Figures 2–3 and supplemental Figures S3–S5.

<a id="kf3"></a>
### KF3: Distinct CAF states and ISG15-associated molecular programs were prioritized using multi-modal data.

Context: Relative enrichment, pseudotime, spatial co-localization and proteomic associations do not prove BRCA1 regulates CAF differentiation through a causal ISG15 pathway.

| Evidence | Value |
| --- | --- |
| CAF state trend | myCAF associated with BRCA1-WT; iCAF associated with BRCA1-MT |
| Spatial observation | 4 BRCA1-WT spatial samples, not a direct genotype-matched spatial comparison |
| Paired proteomics | 9 tumor/adjacent-tissue pairs |
| Marker | ISG15 associated with myCAF enriched signatures |

Source locator: PMC12401915: Results BRCA1-driven CAF reprogramming (s3_6), Multi-scale analysis pinpointed ISG15 (s3_8), ISG15-driven predictor (s3_9); Figures 5–8; Methods high-sensitivity label-free proteomics (s2_11) and supplied DataSheet1.doc Supplementary Methods.

<a id="kf4"></a>
### KF4: The ICI response classifier showed modest retrospective discrimination in treatment cohorts from other cancers.

Context: The held-out subsets come from a mixed-cancer model-selection pipeline; the study states it lacks an ICI-treated TNBC validation cohort.

| Evidence | Value |
| --- | --- |
| Pooled cohort definition | Four cohorts combining to 661 records for 80:20 model development/evaluation |
| Chosen approach | K-nearest neighbors, selected among the models by validation performance |
| Validation ROC AUC | 0.60 (95% CI 0.51–0.69) |
| Testing ROC AUC | 0.61 (95% CI 0.51–0.71) |

Source locator: PMC12401915: Methods Reconstruction of the ICI response predictor (s2_10); Results Construction of an ISG15-driven predictive system (s3_9); Figure 8D–G. Paper text inconsistency: Methods mentions 13 cohorts (n=829); Results mentions 12.

<a id="kf5"></a>
### KF5: High predicted response group correlated with immune-signature and selected survival patterns but does not establish TNBC treatment benefit.

Context: The correlation in METABRIC is not treated ICI response and the TCGA TNBC survival comparison did not reach nominal significance.

| Evidence | Value |
| --- | --- |
| METABRIC overall survival | Reported nominal P<0.05 by predicted response group |
| TCGA TNBC overall survival | P=0.182; not statistically significant |
| TIDE interpretation | Computational exclusion and dysfunction estimates, not observed ICI response |

Source locator: PMC12401915: Results Construction of an ISG15-driven predictive system for ICI response (s3_9); Figure 8H–K; Discussion (s4), paragraph explicitly stating no ICI-treated TNBC validation.

## Study Design & Model Development

### Study profile

- Study Design: Retrospective single-cell TNBC group comparison, spatial reference mapping, bulk expression/network prioritization, paired in-house tissue proteomics and secondary immunotherapy-response classifier development.
- Evidence Type: Human multi-modal observational molecular association with a mixed-cancer retrospective treatment-response model; no experimental causal gene perturbation.
- Population: Eight BRCA1-defined TNBC single-cell specimens; four BRCA1-WT TNBC spatial samples; independently collected 9 paired breast tumor/adjacent tissues; mixed-cancer ICI-response expression cohorts for model training/testing.
- Primary Endpoint: Differences in single-cell and spatially inferred tumor immune/stromal programs across BRCA1 status.
- Secondary Endpoint: ISG15/myCAF association and retrospective response-model discrimination in cross-cancer ICI cohorts.
- External dataset evaluation: No
- Data modalities: TNBC single-cell RNA sequencing, Spatial RNA expression reference mapping, Bulk expression, coexpression and pathway analysis, Paired tissue label-free proteomics, Cross-cancer ICI transcriptomic response modeling

### Model development

- Model Name: ISG15-related ICI-response classifier
- Model Family: K-nearest neighbors selected among evaluated learners
- Development: Mixed-cancer external ICI response cohorts, principal pooled set n=661
- Evaluation: Within-pool 80:20 development split; reported AUC 0.60 validation and 0.61 testing
- Clinical Boundary: No independent treated TNBC response cohort; not suitable as a validated TNBC treatment-selection test.

## What This Study Adds

- Adds exploratory donor-level TNBC immune/stromal states and contextual ISG15 associations with spatial and tissue-proteomic observations.
- Clarifies a crucial translational limitation: modest mixed-cancer response-model discrimination does not validate prediction of ICI benefit in TNBC.

## Evidence Scope

### Supports

- Cell-state and CAF-associated transcriptomic differences between the eight sampled BRCA1 groups.
- Spatially associated ISG15 and myCAF signals within the four BRCA1-WT spatial specimens.
- Retrospective mixed-cancer ICI response model discrimination near AUC 0.60–0.61 under the described analysis.

### Does Not Establish

- Causal BRCA1 control of CAF differentiation or a verified ISG15 immune-response mechanism from the multi-omics associations.
- Independently validated or clinically useful ICI prediction specifically for treated TNBC patients.
- Excellent predictive discrimination, proven chemotherapy/ICI treatment efficacy or a validated treatment-selection cut-off.
- A statistically significant TCGA TNBC overall-survival contrast (reported P=0.182).
- A verified universal count of distinct participants across cell, spatial, paired protein and ICI cohorts.

### Limitations

- The main BRCA1 genotype comparison is only 4 versus 4 tissue donors; cells are not independent participant replicates.
- Spatial transcriptome mapping used four BRCA1-WT cases and therefore is not a genotype-balanced spatial replication.
- ICI cohort descriptions conflict (13 cohorts n=829 in Methods vs 12 in Results), requiring scope-limited cohort reporting.
- The selected KNN internal validation/test AUCs are approximately 0.60–0.61, not high discrimination; use and model selection on validation data constrain performance interpretation.
- No ICI-treated TNBC response cohort directly validates this cross-cancer model.
- The article reports TCGA TNBC overall-survival P=0.182; this comparison is nonsignificant.

<a id="citation-use-cases"></a>
## When This Study Is Useful to Cite

### CU1: What donor-level omics evidence differentiates BRCA1 groups in TNBC?

Eight donor tumors and nested 32,386 cells, limited spatial WT-only series.

Evidence: [KF1](#kf1), [KF2](#kf2), [KF3](#kf3)

### CU2: Can ISG15 be treated as a confirmed causal TNBC immunotherapy mediator?

No; correlative multi-omics without direct functional perturbation.

Evidence: [KF3](#kf3)

### CU3: How much clinical utility is established by the ICI KNN model?

AUC near 0.60 and no directly treated TNBC external validation.

Evidence: [KF4](#kf4), [KF5](#kf5)

<a id="citation-boundaries"></a>
## What This Study Should Not Be Cited to Claim

- Causal BRCA1 control of CAF differentiation or a verified ISG15 immune-response mechanism from the multi-omics associations.
- Independently validated or clinically useful ICI prediction specifically for treated TNBC patients.
- Excellent predictive discrimination, proven chemotherapy/ICI treatment efficacy or a validated treatment-selection cut-off.
- A statistically significant TCGA TNBC overall-survival contrast (reported P=0.182).
- A verified universal count of distinct participants across cell, spatial, paired protein and ICI cohorts.

<a id="evidence-matrix"></a>
## Evidence Matrix

<a id="evidence-b2-doi-10-3389-fonc-2025-1538574-1"></a>
### KF1: The TNBC single-cell series revealed BRCA1-associated differences in annotated tumor immune and stromal states.

- Context: Observed four versus four specimens allow exploratory group comparison; 32,386 cells are nested within eight donors and are not independent patients.
- Finding: The TNBC single-cell series revealed BRCA1-associated differences in annotated tumor immune and stromal states. TNBC samples: 4 BRCA1-MT and 4 BRCA1-WT; QC-retained cells: 32,386; 18,182 mutant-group and 14,204 wild-type-group; Broad annotated lineages: 7 principal populations
- Evidence Level: Observed four versus four specimens allow exploratory group comparison; 32,386 cells are nested within eight donors and are not independent patients.
- Scope: Observed four versus four specimens allow exploratory group comparison; 32,386 cells are nested within eight donors and are not independent patients.
- Source Locator: PMC12401915: Methods Data acquisition and processing (s2_1); Results The single-cell atlas of BRCA1 MT and BRCA1 WT samples (s3_1); Figure 1A–D.
- Evidence refs: [KF1](#kf1)

<a id="evidence-b2-doi-10-3389-fonc-2025-1538574-2"></a>
### KF2: T-cell exhaustion and dendritic/macrophage-associated expression programs differed by BRCA1 group.

- Context: State labels and inferred scores are descriptive transcriptomic signatures; lineage transitions and immunotherapy effectiveness cannot be inferred directly.
- Finding: T-cell exhaustion and dendritic/macrophage-associated expression programs differed by BRCA1 group. Comparison: BRCA1-WT versus BRCA1-MT single-cell specimens; CD4/CD8 states: Treg, Tex and other computationally annotated subsets; Intercellular readouts: Pseudotime and inferred spatial signaling, not direct lineage tracing
- Evidence Level: State labels and inferred scores are descriptive transcriptomic signatures; lineage transitions and immunotherapy effectiveness cannot be inferred directly.
- Scope: State labels and inferred scores are descriptive transcriptomic signatures; lineage transitions and immunotherapy effectiveness cannot be inferred directly.
- Source Locator: PMC12401915: Results T cells in BRCA1-WT specimens (s3_2); Activated TAMs and tolerogenic DCs (s3_3); Figures 2–3 and supplemental Figures S3–S5.
- Evidence refs: [KF2](#kf2)

<a id="evidence-b2-doi-10-3389-fonc-2025-1538574-3"></a>
### KF3: Distinct CAF states and ISG15-associated molecular programs were prioritized using multi-modal data.

- Context: Relative enrichment, pseudotime, spatial co-localization and proteomic associations do not prove BRCA1 regulates CAF differentiation through a causal ISG15 pathway.
- Finding: Distinct CAF states and ISG15-associated molecular programs were prioritized using multi-modal data. CAF state trend: myCAF associated with BRCA1-WT; iCAF associated with BRCA1-MT; Spatial observation: 4 BRCA1-WT spatial samples, not a direct genotype-matched spatial comparison; Paired proteomics: 9 tumor/adjacent-tissue pairs; Marker: ISG15 associated with myCAF enriched signatures
- Evidence Level: Relative enrichment, pseudotime, spatial co-localization and proteomic associations do not prove BRCA1 regulates CAF differentiation through a causal ISG15 pathway.
- Scope: Relative enrichment, pseudotime, spatial co-localization and proteomic associations do not prove BRCA1 regulates CAF differentiation through a causal ISG15 pathway.
- Source Locator: PMC12401915: Results BRCA1-driven CAF reprogramming (s3_6), Multi-scale analysis pinpointed ISG15 (s3_8), ISG15-driven predictor (s3_9); Figures 5–8; Methods high-sensitivity label-free proteomics (s2_11) and supplied DataSheet1.doc Supplementary Methods.
- Evidence refs: [KF3](#kf3)

<a id="evidence-b2-doi-10-3389-fonc-2025-1538574-4"></a>
### KF4: The ICI response classifier showed modest retrospective discrimination in treatment cohorts from other cancers.

- Context: The held-out subsets come from a mixed-cancer model-selection pipeline; the study states it lacks an ICI-treated TNBC validation cohort.
- Finding: The ICI response classifier showed modest retrospective discrimination in treatment cohorts from other cancers. Pooled cohort definition: Four cohorts combining to 661 records for 80:20 model development/evaluation; Chosen approach: K-nearest neighbors, selected among the models by validation performance; Validation ROC AUC: 0.60 (95% CI 0.51–0.69); Testing ROC AUC: 0.61 (95% CI 0.51–0.71)
- Evidence Level: The held-out subsets come from a mixed-cancer model-selection pipeline; the study states it lacks an ICI-treated TNBC validation cohort.
- Scope: The held-out subsets come from a mixed-cancer model-selection pipeline; the study states it lacks an ICI-treated TNBC validation cohort.
- Source Locator: PMC12401915: Methods Reconstruction of the ICI response predictor (s2_10); Results Construction of an ISG15-driven predictive system (s3_9); Figure 8D–G. Paper text inconsistency: Methods mentions 13 cohorts (n=829); Results mentions 12.
- Evidence refs: [KF4](#kf4)

<a id="evidence-b2-doi-10-3389-fonc-2025-1538574-5"></a>
### KF5: High predicted response group correlated with immune-signature and selected survival patterns but does not establish TNBC treatment benefit.

- Context: The correlation in METABRIC is not treated ICI response and the TCGA TNBC survival comparison did not reach nominal significance.
- Finding: High predicted response group correlated with immune-signature and selected survival patterns but does not establish TNBC treatment benefit. METABRIC overall survival: Reported nominal P<0.05 by predicted response group; TCGA TNBC overall survival: P=0.182; not statistically significant; TIDE interpretation: Computational exclusion and dysfunction estimates, not observed ICI response
- Evidence Level: The correlation in METABRIC is not treated ICI response and the TCGA TNBC survival comparison did not reach nominal significance.
- Scope: The correlation in METABRIC is not treated ICI response and the TCGA TNBC survival comparison did not reach nominal significance.
- Source Locator: PMC12401915: Results Construction of an ISG15-driven predictive system for ICI response (s3_9); Figure 8H–K; Discussion (s4), paragraph explicitly stating no ICI-treated TNBC validation.
- Evidence refs: [KF5](#kf5)

## Q&A

### How many single-cell patients were studied and how many cells were retained?

Eight donor tumor specimens were analyzed (four BRCA1-MT, four BRCA1-WT), generating 32,386 quality-controlled cells; cell counts must not be reported as independent participant counts.

Evidence: KF1

### Was there a genotype-matched spatial comparison?

Only four BRCA1-WT spatial transcriptomic samples were described. The spatial analysis cannot independently validate BRCA1-MT versus WT spatial differences in a matched specimen design.

Evidence: KF3

### What evidence nominates ISG15?

ISG15 was associated with myCAF-related modules, spatial expression and paired proteomic observations. These are multi-modal correlations and do not directly prove its functional mediation.

Evidence: KF3

### How well did the ICI response model discriminate?

The selected KNN classifier reported AUC 0.60 (95% CI 0.51–0.69) for validation and 0.61 (95% CI 0.51–0.71) for testing in the external-treatment source data.

Evidence: KF4

### Was the ICI model validated in treated TNBC patients?

No. The Discussion explicitly identifies the lack of validation in ICI-treated TNBC cohorts; it is inappropriate to claim established TNBC treatment selection.

Evidence: KF4, KF5

### Did the model-selected groups have significantly different TCGA TNBC overall survival?

No. The reported TCGA TNBC overall survival comparison was P=0.182, not statistically significant. A nominal METABRIC comparison does not establish ICI clinical benefit.

Evidence: KF5

### How many ICI cohorts did the authors use?

The article is inconsistent: Methods describes 13 pretreatment ICI cohorts (829 records) whereas Results mentions 12 cohorts. Four cohorts pooled as 661 records for the principal development split can be identified; the other cohort count is not silently harmonized.

Evidence: KF4

## Concepts & Entities

### Disease And Cells

Triple-negative breast cancer, BRCA1 mutation status, Cancer-associated fibroblasts

### Omics And Biomarkers

Single-cell and spatial transcriptomics, Paired tissue proteomics, ISG15

### Prediction Scope

Cross-cancer KNN response model, AUC approximately 0.61, No treated TNBC validation

## Related Research

- [Deciphering the tumor immune microenvironment from a multidimensional omics perspective: insight into next-generation CAR-T cell immunotherapy and beyond](https://drgezhang.com/papers/time-cart.html) — Narrative TIME/CAR-T review that provides research context for immunosuppressive cell states but does not validate the TNBC ISG15 model. (DOI: 10.1186/s12943-024-02047-2)
- [Novel post-translational modification learning signature reveals B4GALT2 as an immune exclusion regulator in lung adenocarcinoma](https://drgezhang.com/papers/b4galt2.html) — A distinct LUAD PTMLS/B4GALT2 immune-exclusion study, related by computational and immune-state themes but not TNBC-specific validation. (DOI: 10.1136/jitc-2024-010787)

## Publication & Provenance

- DOI: https://doi.org/10.3389/fonc.2025.1538574
- Doi Url: https://doi.org/10.3389/fonc.2025.1538574
- Publisher: https://www.frontiersin.org/journals/oncology/articles/10.3389/fonc.2025.1538574/full
- Pmc Url: https://pmc.ncbi.nlm.nih.gov/articles/PMC12401915/
- PMCID: PMC12401915
- License: Article-level CC BY 4.0 in PMC/Frontiers. This evidence page is original factual synthesis without copy-pasting the study full text.
- License Url: https://creativecommons.org/licenses/by/4.0/
- Version Of Record: Frontiers in Oncology 2025, DOI 10.3389/fonc.2025.1538574; published 19 August 2025.
- Source Artifacts: PMC VOR/JATS; supplemental DataSheet1.doc methods read with antiword, DataSheet2.docx figures, Tables S1–S4, and supplied publisher/PMC figures.
- Numerical Discrepancy: Main Methods reports 13 ICI pretreatment cohorts (n=829), Results states 12 cohorts; no unsupported cross-series total is accepted.
- Clinical Boundary: Cross-cancer ICI model tested in retrospective data but no treated TNBC response validation; only modest AUC approximately 0.60–0.61.

## Evidence-page notice

This evidence page distinguishes donor specimens, nested cells, spatial spots, paired proteins and ICI cohorts. The KNN model has modest discrimination and lacks treated TNBC validation; do not cite it as demonstrated ICI treatment benefit.

## Links

- DOI: https://doi.org/10.3389/fonc.2025.1538574
- HTML page: https://drgezhang.com/papers/doi-10-3389-fonc-2025-1538574.html
- Google Scholar query: https://scholar.google.com/scholar?q=%22Resolving%20tumor%20microenvironment%20heterogeneity%20to%20forecast%20immunotherapy%20response%20in%20triple-negative%20breast%20cancer%20through%20multi-scale%20analysis%22
- ORCID: https://orcid.org/0000-0002-3116-3246