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Breast cancer stands as a formidable global health challenge for women. While neoantigens exhibit efficacy in activating T cells specific to cancer and instigating anti-tumor immune responses, the accuracy of neoantigen prediction remains suboptimal. In this study, we identified neoantigens from the patient-derived breast cancer cells, PC-B-142CA and PC-B-148CA cells, utilizing whole-genome and RNA sequencing. The pVAC-Seq pipeline was employed, with minor modification incorporating criteria (1) binding affinity of mutant (MT) peptide with HLA (IC50 MT) ≤ 500 nm in 3 of 5 algorithms and (2) IC50 wild type (WT)/MT > 1. Sequencing results unveiled 2513 and 3490 somatic mutations, and 646 and 652 non-synonymous mutations in PC-B-142CA and PC-B-148CA, respectively. We selected the top 3 neoantigens to perform molecular dynamic simulation and synthesized 9-12 amino acid neoantigen peptides, which were then pulsed onto healthy donor peripheral blood mononuclear cells (PBMCs). Results demonstrated that T cells activated by ADGRL1E274K, PARP1E619K, and SEC14L2R43Q peptides identified from PC-B-142CA exhibited significantly increased production of interferon-gamma (IFN-γ), while PARP1E619K and SEC14L2R43Q peptides induced the expression of CD107a on T cells. The % tumor cell lysis was notably enhanced by T cells activated with MT peptides across all three healthy donors. Moreover, ALKBH6V83M and GAAI823T peptides from PC-B-148CA remarkably stimulated IFN-γ- and CD107a-positive T cells, displaying high cell-killing activity against target cancer cells. In summary, our findings underscore the successful identification of neoantigens with anti-tumor T cell functions and highlight the potential of personalized neoantigens as a promising avenue for breast cancer treatment.
Assuntos
Neoplasias da Mama , Feminino , Humanos , Leucócitos Mononucleares , Linfócitos T , Algoritmos , AnticorposRESUMO
BACKGROUND: Triple-negative breast cancer (TNBC), known for its aggressiveness and limited treatment options, presents a significant challenge. Adoptive cell transfer, involving the ex vivo generation of antigen-specific T cells from peripheral blood mononuclear cells (PBMCs), emerges as a promising approach. The overexpression of mesothelin (MSLN) and nucleolin (NCL) in TNBC samples underscores their potential as targets for T cell therapy. This study explored the efficacy of multi-peptide pulsing of PBMCs to generate MSLN/NCL-specific T cells targeting MSLN+/NCL+ TNBC cells. METHODS: TNBC patient samples were confirmed for both MSLN and NCL expression via immunohistochemistry. Synthesized MSLN and NCL peptides were combined and administered to activate PBMCs from healthy donors. The cancer-killing ability of the resultant T cells was assessed using crystal violet staining, and their subtypes and cytotoxic cytokines were characterized through flow cytometry and cytokine bead array. RESULTS: Findings showed that 85.3% (127/149) of TNBC cases were positive for either MSLN or NCL, or both; with single positivity rates for MSLN and NCL of 14.1% and 28.9%, respectively. MSLN and NCL peptides, with high binding affinity for HLA-A*02, were combined and introduced to activated PBMCs from healthy donors. The co-pulsed PBMCs significantly induced TEM and TEMRA CD3+/CD8+ T cells and IFN-γ production, compared to single-peptide pulsed or unpulsed conditions. Notably, MSLN/NCL-specific T cells successfully induced cell death in MSLN+/NCL+ MDA-MB-231 cells, releasing key cytotoxic factors such as perforin, granzymes A and B, Fas ligand, IFN-γ, and granulysin. CONCLUSIONS: These findings serve as a proof-of-concept for using multiple immunogenic peptides as a novel therapeutic approach in TNBC patients.
Assuntos
Mesotelina , Nucleolina , Peptídeos , Linfócitos T , Neoplasias de Mama Triplo Negativas , Adulto , Feminino , Humanos , Pessoa de Meia-Idade , Linhagem Celular Tumoral , Citocinas/metabolismo , Imunoterapia Adotiva/métodos , Leucócitos Mononucleares/imunologia , Linfócitos T/imunologia , Neoplasias de Mama Triplo Negativas/imunologiaRESUMO
Background: Tumoral hypoxia is associated with aggressiveness in many cancers including breast cancer. However, measuring hypoxia is complicated. Carbonic anhydrase IX (CAIX) is a reliable endogenous marker of hypoxia under the control of the master regulator hypoxia-inducible factor-1α (HIF-1α). The expression of CAIX is associated with poor prognosis in many solid malignancies; however, its role in breast cancer remains controversial. Methods: The present study performed a meta-analysis to evaluate the correlation between CAIX expression and disease-free survival (DFS) and overall survival (OS) in breast cancer. Results: A total of 2,120 publications from EMBASE, PubMed, Cochrane, and Scopus were screened. Of these 2,120 publications, 272 full texts were reviewed, and 27 articles were included in the meta-analysis. High CAIX was significantly associated with poor DFS (HR = 1.70, 95% CI = 1.39-2.07, p < 0.00001) and OS (HR = 2.02, 95% CI 1.40-2.91, p = 0.0002) in patients with breast cancer. When stratified by subtype, the high CAIX group was clearly associated with shorter DFS (HR = 2.09, 95% CI =1.11-3.92, p = 0.02) and OS (HR = 2.50, 95% CI =1.53-4.07, p = 0.0002) in TNBC and shorter DFS in ER+ breast cancer (HR = 1.81 95% CI =1.38-2.36, p < 0.0001). Conclusion: High CAIX expression is a negative prognostic marker of breast cancer regardless of the subtypes.
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Nucleolin (NCL) is a multifunctional protein expressed in the nucleus, cytoplasm, and cell membrane. Overexpression of NCL has a controversial role as a poor prognostic marker in cancers. In this study, a meta-analysis was performed to evaluate the prognostic value of NCL in different subcellular localizations (cytoplasmic (CyNCL) and nuclear (NuNCL)) across a range of cancers. PubMed was searched for relevant publications. Data were extracted and analyzed from 12 studies involving 1221 patients with eight cancer types. The results revealed high total NCL was significantly associated with poor overall survival (OS) (HR = 2.85 (1.94, 4.91), p < 0.00001, I2 = 59%) and short disease-free survival (DFS) (HR = 3.57 (2.76, 4.62), p < 0.00001, I2 = 2%). High CyNCL was significantly associated with poor OS (HR = 4.32 (3.01, 6.19), p < 0.00001, I2 = 0%) and short DFS (HR = 3.00 (2.17, 4.15), p < 0.00001, I2 = 0%). In contrast, high NuNCL correlated with increased patient OS (HR = 0.42 (0.20, 0.86), p = 0.02, I2 = 66%), with no significant correlation to DFS observed (HR = 0.46 (0.19, 1.14), p = 0.09, I2 = 57%). This study supports the role of subcellular NCL as a poor prognostic cancer biomarker.
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Neoplasias , Fosfoproteínas , Intervalo Livre de Doença , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/genética , NucleolinaRESUMO
Breast cancer cell lines are widely used as an in vitro system with which to study the mechanisms underlying biological and chemotherapeutic resistance. In the present study, two novel breast cancer cell lines designated as PCB142CA and PCB148CA were successfully established from HER2positive and triplenegative (TN) breast cancer tissues. The cell lines were characterized by cytokeratin (CK), αsmooth muscle actin (αSMA), fibroblastactivation protein (FAP) and programmed deathligand 1 (PDL1). Cell proliferation was assessed using a colony formation assay, an MTS assay, 3dimensional (3D) spheroid and 3D organoid models. Wound healing and Transwell migration assays were used to explore the cell migration capability. The responses to doxorubicin (DOX) and paclitaxel (PTX) were evaluated by 3D spheroids. The results showed that the PCB142CA and PCB148CA cell lines were αSMAnegative, FAPnegative, CKpositive and PDL1positive. Both cell lines were adherent with the ability of 3Dmulticellular spheroid and organoid formations; invadopodia were found in the spheroids/organoids of only PCB148CA. PCB142CA had a faster proliferative but lower metastatic rate compared to PCB148CA. Compared to MDAMB231, a commercial TN breast cancer cell line, PCB148CA had a similar CD44+/CD24 stemness property (96.90%), whereas only 8.75% were found in PCB142CA. The mutations of BRCA1/2, KIT, PIK3CA, SMAD4, and TP53 were found in PCB142CA cells related to the resistance of several drugs, whereas PCB148CA had mutated BRCA2, NRAS and TP53. In conclusion, PCB142CA can serve as a novel HER2positive breast cancer cell line for drug resistance studies; while PCB148CA is a novel TN breast cancer cell line suitable for metastatic and stemnessrelated properties.