Abstract P10: Early Immunosuppressive Landscape Shaped by BRCA1/p53 Loss in Early Pre-Malignant Mammary Glands Permits Tumor Development

TLSB-09 TARGETED P16 KNOCKOUT CONFERS AN IMMUNOSUPPRESSIVE MICROENVIRONMENT IN PRECLINICAL MODELS OF TRIPLE NEGATIVE BREAST CANCER BRAIN METASTASIS

Abstract Loss of the 9p21 chromosomal locus, which contains CDKN2A, has been associated with non-response to immune checkpoint inhibition (ICI) across multiple human tumor histologies, yet the underlying mechanisms remain elusive. Given the high frequency of CDK pathway alterations in human brain metastases (BM), especially those arising from triple negative breast cancer (TNBC), we sought to explore the relationship between loss of p16, a CDKN2A isoform, and ICI resistance associated with 9p21 loss. We used CRISPR-Cas9 to generate an isogenic p16-knockout derivative (EMT6 sgRNA3) from ICI-sensitive murine TNBC cell line EMT6, as well as a non-targeting control (EMT6 sgNT). With these cell lines, we established an in vivo model in BALB/c mice to assess intracranial response to anti-PD-1 treatment. Preliminary results revealed a trend toward diminished intracranial ICI efficacy in mice bearing EMT6 sgRNA3 tumors compared to EMT6 sgNT controls (n = 10/group, p=0.08). To validate this finding, we generated ex vivo organotypic spheroids (OTS) from intracranial tumors at survival endpoint and assessed anti-PD-1-induced cell death by lactate dehydrogenase release. Compared to IgG control (10 μg/mL), anti-PD-1 (10 μg/mL) treatment produced a significant increase in cytotoxicity in EMT6 sgNT OTS but not EMT6 sgRNA3 OTS (p=0.001 vs. p=0.76, respectively) after 72 hours. To interrogate the mechanistic basis of these findings, we performed bulk RNAseq on intracranial tumors (n = 4/group), which revealed a significant decrease in expression of chemokines (Cxcl1, Cxcl10, Cxcl11) and immune-related genes (Cd274, Vcam1, Il2ra) in EMT6 sgRNA3 tumors, suggesting that p16 loss is associated with an immunosuppressive microenvironment characterized by decreased innate immune signaling and immune cell recruitment. These findings suggest a role for p16 loss as a candidate functional driver of ICI resistance associated with 9p21 loss in TNBC-BM and warrant further investigation given the need to nominate biomarkers for ICI response to inform patient care.

KRAS mutations as architects of the tumor immune microenvironment: implications for combination therapies

Oncogenic KRAS mutations rank among the most prevalent driver alterations in human malignancies, reaching near-universal frequency (~98%) in pancreatic ductal adenocarcinoma (PDAC) and high prevalence in colorectal cancer (CRC, ~52%) and lung adenocarcinoma (LAC, ~32%). Beyond their canonical roles in promoting cell-intrinsic proliferation and survival through the MAPK/ERK and PI3K/AKT cascades, KRAS mutations actively sculpt a profoundly immunosuppressive tumor microenvironment (TME), which constitutes a major barrier to both targeted therapy and immunotherapy. Through coordinated programs encompassing inflammatory cytokine secretion, downregulation of antigen presentation machinery, tumor-associated macrophage (TAM) reprogramming, myeloid-derived suppressor cell (MDSC) expansion, and PD-L1 upregulation, KRAS-mutant tumors establish robust immune exclusion. These programs are further stratified by co-mutations in STK11 , KEAP1 , and TP53 , which define distinct immune phenotypes ranging from inflamed to profoundly immune-excluded “cold” tumors. The recent approval of covalent KRAS G12C inhibitors, sotorasib and adagrasib, has revealed that targeted KRAS blockade can remodel the TME toward an immunostimulatory state, providing a mechanistic rationale for combining KRAS-directed agents with immune checkpoint blockade, STING agonists, and neoantigen vaccines. This mini-review synthesizes the current knowledge of KRAS-immune crosstalk, highlights existing controversies and research gaps, and evaluates emerging combination strategies designed to convert immune exclusion into durable anti-tumor immunity.

Macrophage heterogeneity in breast cancer microenvironment and development of a prognostic model: exploring TFF1 as a novel therapeutic target.

BACKGROUNDS Breast cancer (BRCA) represents the most prevalent malignancy globally, with projections indicating 3.2 million new cases anticipated by 2050. Current treatment modalities, encompassing surgical intervention, chemotherapy, and immunotherapy, are markedly hindered by treatment resistance and the inherent complexity of BRCA, thereby impeding effective disease management. Consequently, this study is designed to elucidate cellular heterogeneity within the tumor microenvironment (TME) and to identify prospective therapeutic targets, thus enabling individualized treatment approaches for individuals with BRCA, which remains crucial. METHODS GEO and TCGA databases were the sources of all analytical data employed in this investigation. Single-cell RNA sequencing data were employed to identify macrophage-associated genes in BRCA, followed by the development of a machine-learning-based prognostic model (PM) integrating data from TCGA and GEO databases. This model stratifies individuals into cohorts of either low or high risk, enabling a disparity evaluation in survival outcomes and tumor immune microenvironment characteristics. Gene co-expression patterns were examined through an analysis of gene co-expression network weighted by high dimension for screening pivotal central genes implicated in tumor immunity. Clinical PMs were subsequently constructed utilizing machine learning algorithms, with validation performed on training and test sets. Furthermore, XGBoost and LightGBM machine learning algorithms were implemented to pinpoint potential biomarkers. Ultimately, validation of these biomarkers was conducted through ELISA, CCK-8 assay, flow cytometry, Western blotting, and immunoprecipitation assays. RESULTS Findings indicated a pronounced elevation of TAMs in BRCA patients, with their phenotypic attributes exhibiting a strong correlation with prognosis. The developed clinical PM demonstrated high accuracy and robust predictive capability concerning survival rates of 1, 3, or 5 years. The comprehensive evaluation determined that high-risk cohort's immune microenvironment exhibited a greater inclination toward immune suppression, whereas the low-risk cohort was markedly predisposed to anti-tumor immune responses. Notably, the trifolium factor 1 (TFF1) gene was identified as a key determinant, with its overexpression being markedly associated with tumor invasiveness and immune evasion. CONCLUSIONS The study underscores cellular heterogeneity inherent in BRCA TME, while PM, predicated on macrophage subpopulation-related genes, offers a novel approach for risk stratification and personalized therapeutic interventions for BRCA patients. Moreover, TFF1 has been established as a prospective therapeutic target, yielding important directions for developing specific treatment interventions.

Immune evasion in locally advanced mismatch repair-deficient microsatellite instability-high colorectal cancer: Reduced T-cell infiltration and upregulation of epithelial IDO1 expression

ABSTRACT Early-stage colorectal cancer (CRC) with confirmed microsatellite instability generally has a favorable prognosis associated with pronounced immune infiltration. However, some patients still develop metastases. The underlying mechanisms, particularly those related to the tumor immune microenvironment, remain incompletely understood. The study included tissue samples from 217 patients with dMMR/MSI-H CRC, comprising 89 stage III/IV and 128 stage I/II cases. Tissue microarrays and immunohistochemical analyzes were performed for all cases. We evaluated immune markers identifying T cells, B cells, dendritic cells, natural killer cells, macrophages, immunosuppressive markers, and immune checkpoint targets in epithelial and stromal compartments, and additionally performed a cohort-derived immune infiltration score (IIS). The survival analysis assessed the prognostic impact of immune markers stratified by tumor stage. Stage I/II dMMR/MSI-H CRCs showed significantly higher CD3⁺ T-cell and natural killer cell levels, higher IIS metrics across all regions, and higher CD4⁺ T helper cell levels in the stroma. Stage III/IV cases exhibited increased epithelial expression of indoleamine 2,3-dioxygenase 1. Given the limited number of stage IV patients, an additional stage III vs. stage I/II comparison was performed, revealing that these immune differences were already evident at the level of nodal progression. In addition, CD3⁺, CD4⁺, and CD8⁺ T cells and the IIS showed varying prognostic associations across tumor stages. These findings suggest that the overall immunogenicity and prognostic relevance of immune markers in dMMR/MSI-H CRC depend on tumor stage. Immune profiling could be used for early-stage patient stratification and also suggests the potential benefit of early immunotherapeutic intervention.

Imbalanced p53-RB signaling triggers cGAS-STING-mediated immune activation in early oral cancer development.

Tumor-Intrinsic DNA Damage Signaling Is Associated with MHC-I Expression and CD8 Cytotoxic T-Cell Engagement in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is characterized by marked immune microenvironment heterogeneity and variable chemotherapy response, yet the epithelial transcriptional programs governing cytotoxic immune activation remain poorly understood. We performed an exploratory, integrative analysis using single-cell RNA sequencing of 31,962 cells from eight TNBC patients operationally stratified into Good and Bad Prognosis groups based on pathological lymphoid infiltration, a discovery grouping subsequently validated against pathological complete response (pCR) in three independent bulk RNA-seq cohorts. This analysis identified four epithelial transcriptional states. The G5 DNA damage subpopulation—predominantly restricted to Good Prognosis tumors (29.2% vs. 0%)—and the G4 Metabolism subpopulation—2.4-fold enriched in Bad Prognosis—were the primary prognostic signatures. Machine learning validation using nested leave-one-cohort-out (LOCO) cross-validation across 614 samples demonstrated that G4 + G5 raw genes with random forest yielded the largest observed mean AUC of 0.653, though these results are exploratory and do not establish a validated clinical classifier. CellChat ligand–receptor interaction analysis revealed that G5 DNA-damage epithelial cells are the dominant immune activators in Good Prognosis TNBC, predominantly engaging CD8 cytotoxic T cells through MHC-I antigen presentation via HLA-A/B/C/E/F → CD8A/CD8B interactions, the highest-probability signaling pathway identified. Spatial transcriptomics independently validated significantly higher DNA damage and CD8 T-cell scores in Good Prognosis tissue. Together, these exploratory findings suggest a framework in which tumor-intrinsic DNA damage signaling is associated with MHC-I antigen presentation upregulation and CD8 cytotoxic T-cell engagement, supporting further investigation of this axis and its potential implications for combining DNA-damaging chemotherapy with immune checkpoint blockade in TNBC.