Stimulating T cell responses against patient-derived breast cancer cells with neoantigen peptide-loaded peripheral blood mononuclear cells.

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.

Carbonic anhydrase IX-related tumoral hypoxia predicts worse prognosis in breast cancer: A systematic review and meta-analysis.

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.

Cellular localization of nucleolin determines the prognosis in cancers: a meta-analysis.

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.

Establishment and characterization of novel highly aggressive HER2­positive and triple­negative breast cancer cell lines.

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.