Immunotherapy In Triple-negative Breast Cancer Research Articles

Click Chemistry-Mediated Polymannose Surface-Engineering of Natural Killer Cells for Immunotherapy of Triple-Negative Breast Cancer.

Natural killer (NK) cells, serve as the frontline defense of the immune system, and are capable of surveilling and eliminating tumor cells. Their significance in tumor immunotherapy has garnered considerable attention in recent years. However, the absence of specific receptor-ligand interactions between NK cells and tumor cells hampers their selectivity, thereby limiting the therapeutic effectiveness of NK cell-based tumor immunotherapy. Herein, this work constructs polymannose-engineered NK (pM-NK) cells via metabolic glycoengineering and copper-free click chemistry. Polymannose containing dibenzocyclooctyne terminal groups (pM-DBCO) is synthesized and covalently modified on the surface of azido-labeled NK cells. Compared to the untreated NK cells, the interactions between pM-NK cells and MDA-MB-231 cells, a breast tumor cell line with overexpression of mannose receptors (MRs), are significantly increased, and lead to significantly enhanced killing efficacy. Consequently, intravenous administration of pM-NK cells will effectively inhibit the tumor growth and will prolong the survival of mice bearing MDA-MB-231 tumors. Thus, this work presents a novel strategy for tumor-targeting NK cell-based tumor immunotherapy.

The potent potential of MFAP2 in prognosis and immunotherapy of triple-negative breast cancer

BackgroundsMicrofibril-associated protein 2 (MFAP2) is a protein presenting in the extracellular matrix that governs the activity of microfibrils through its interaction with fibrillin. While the involvement of MFAP2 in metabolic disorders has been documented, its expression and prognostic significance in triple-negative breast cancer (TNBC) remain unexplored.MethodsWe acquired datasets pertaining to breast cancer (BC) from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Next, a Venn diagram was used to identify the differentially expressed genes (DEGs). The DEGs were used to perform Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein–protein interaction (PPI), immune and survival analysis. The expressions of MFAP2, PD-1 and PD-L1 were examined by immunohistochemistry and western blot and their relationship with clinical pathological parameters were analyzed by clinical specimen samples from patients with TNBC. Tumor Immune Estimation Resource (TIMER, https://cistrome.shinyapps.io/timer/) was adopted to calculate the immune infiltration level of TNBC. The link between gene expression and tumor mutational burden (TMB) was described using Spearman’s correlation analysis.ResultsWe identified 66 differentially expressed genes (DEGs) that were up-regulated. Among these DEGs, MFAP2 was found to be overexpressed in TNBC and was associated with a lower probability of survival. This finding was confirmed through the use of immunohistochemistry and western blot techniques. Additionally, MFAP2 was found to be related to various pathological parameters in TNBC patients. Mechanistically, gene set enrichment analysis (GSEA) revealed that MFAP2 primarily influenced cellular biological behavior in terms of epithelial mesenchymal transition, glycolysis, and apical junction. Notably, MFAP2 expression was positively correlated with the abundance of macrophages, while a negative correlation was observed with the abundance of B cells, CD4 + T cells, CD8 + T cells, neutrophils and dendritic cells through immune analysis. Furthermore, it was observed that MFAP2 displayed a negative correlation not only with tumor mutational burden (TMB), a recognized biomarker for PD-1/PD-L1 immunotherapy, but also with PD-L1 in samples of TNBC.ConclusionMFAP2 may be an important prognostic biomarker for TNBC, as well as a viable target for immunotherapy in this disease.

Nanoparticle-Mediated Immunotherapy in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype with the worst prognosis and highest recurrence rates. The treatment choices are limited due to the scarcity of endocrine and HER2 targets, except for chemotherapy. However, the side effects of chemotherapy restrict its long-term usage. Immunotherapy shows potential as a promising therapeutic strategy, such as inducing immunogenic cell death, immune checkpoint therapy, and immune adjuvant therapy. Nanotechnology offers unique advantages in the field of immunotherapy, such as improved delivery and targeted release of immunotherapeutic agents and enhanced bioavailability of immunomodulators. As well as the potential for combination therapy synergistically enhanced by nanocarriers. Nanoparticles-based combined application of multiple immunotherapies is designed to take the tactics of enhancing immunogenicity and reversing immunosuppression. Moreover, the increasing abundance of biomedical materials holds more promise for the development of this field. This review summarizes the advances in the field of nanoparticle-mediated immunotherapy in terms of both immune strategies for treatment and the development of biomaterials and presents challenges and hopes for the future.

Role of ICAM-1 in triple-negative breast cancer.

Intercellular adhesion molecule-1 (ICAM-1) is related to the occurrence and development of a variety of tumors. However, the role of ICAM-1 in the regulation of growth, metastasis, and clinical prognosis of the specific molecular subtypes of breast cancer, triple-negative breast cancer (TNBC), remains to be elucidated. This study explored the role of ICAM-1 in breast cancer and its triple-negative subtypes by systematic bioinformatics methods. The results showed that the expression of ICAM-1 in breast cancer tissues was significantly higher than that in normal tissues, especially in TNBC subtypes. In breast cancer, ICAM-1 mainly activates pathways related to apoptosis and epithelial-mesenchymal transition, while its overexpression in TNBC is associated with inflammatory response, apoptosis, and other processes. TNBC patients displaying higher ICAM-1 expression demonstrate enhanced responses to immunotherapy. High ICAM-1 expression is sensitive to drugs targeting tumor cell proliferation, apoptosis, and angiogenesis. In conclusion, breast cancer is characterized by significantly high expression of ICAM-1, with TNBC subtypes expressing ICAM-1 at much higher levels than other subtypes. The diagnosis, prognosis, development, distant metastases, and immunotherapy of TNBC are correlated with high expression of ICAM-1. This research provides available data for the further study of the diagnosis and treatment of TNBC.

Artificial intelligence enhances whole-slide interpretation ofPD-L1 CPS in triple-negative breast cancer: Amulti-institutional ring study.

Evaluation of the programmed cell death ligand-1 (PD-L1) combined positive score (CPS) is vital to predict the efficacy of the immunotherapy in triple-negative breast cancer (TNBC), but pathologists show substantial variability in the consistency and accuracy of the interpretation. It is of great importance to establish an objective and effective method which is highly repeatable. We proposed a model in a deep learning-based framework, which at the patch level incorporated cell analysis and tissue region analysis, followed by the whole-slide level fusion of patch results. Three rounds of ring studies (RSs) were conducted. Twenty-one pathologists of different levels from four institutions evaluated the PD-L1 CPS in TNBC specimens as continuous scores by visual assessment and our artificial intelligence (AI)-assisted method. In the visual assessment, the interpretation results of PD-L1 (Dako 22C3) CPS by different levels of pathologists have significant differences and showed weak consistency. Using AI-assisted interpretation, there were no significant differences between all pathologists (P = 0.43), and the intraclass correlation coefficient (ICC) value was increased from 0.618 [95% confidence interval (CI) = 0.524-0.719] to 0.931 (95% CI = 0.902-0.955). The accuracy of interpretation result is further improved to 0.919 (95% CI = 0.886-0.947). Acceptance of AI results by junior pathologists was the highest among all levels, and 80% of the AI results were accepted overall. With the help of the AI-assisted diagnostic method, different levels of pathologists achieved excellent consistency and repeatability in the interpretation of PD-L1 (Dako 22C3) CPS. Our AI-assisted diagnostic approach was proved to strengthen the consistency and repeatability in clinical practice.

CXCR6 expression correlates with radiotherapy response and immune context in triple-negative breast cancer (Experimental Studies)

Background: The chemokine receptor CXCR6 is critical for sustained tumor control mediated by CD8+ cytotoxic T cells (CTLs) in tumors. Previous studies have shown that ionizing radiation induces an inflamed immune contexture by upregulating CXCR6. However, the clinical significance of CXCR6 expression in triple-negative breast cancer (TNBC) and its correlation with radiotherapy remains unknown. This study aimed to clarify the prognostic value of CXCR6 and its role in the breast tumor microenvironment (TME). Methods: The mRNA and protein expression of CXCR6 in human triple-negative breast cancer and their association with survival were analyzed. The role of CXCR6 in the immune context was investigated using a combination of single-cell RNA sequencing, general transcriptome sequencing data and fluorescence-based multiplex immunohistochemistry (mIHC) techniques. Results: Elevated CXCR6 expression correlated with better clinical outcomes and superior response to adjuvant radiotherapy and immunotherapy in TNBC. CXCR6 fostered an immunostimulatory microenvironment characterized by upregulated cytotoxic markers. We also found that CXCR6 plays a crucial role in regulating the differentiation of CD8+ T cells and the intercellular communication of immune cell subtypes, shaping the anti-tumor microenvironment. Conclusions: This study highlights the emerging role of CXCR6 in shaping the antitumor immune microenvironment, and targeting CXCR6 may be a promising strategy for improving the effectiveness of radiotherapy and immunotherapy in TNBC.

Abstract PO1-16-03: GDP-mannose enhances the efficacy of PARP inhibitors and immunotherapy in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) patients with high HRD scores benefit from poly (ADP-ribose) polymerase (PARP) inhibitors, while those with low HRD scores still lack therapeutic options. Homologous recombination deficiency (HRD) induction is an effective strategy to broaden the indications of PARP inhibitors. Metabolic reprogramming is a critical feature of TNBC. In this study, we aimed to explore novel metabolic biomarkers for HRD and new strategy for sensitizing PARP inhibitors. Methods: We utilized TNBC metabolomics to systematically evaluate metabolites that were correlated with HRD. A crucial metabolite, GDP-mannose (GDP-M), that impedes homologous recombination repair (HR) and sensitizes PARP inhibitors was identified and functionally validated. We further explored the detailed mechanism and proposed a potential treatment strategy utilizing GDP-M for TNBC in preclinical models. Results: Systematic metabolomic analysis revealed that GDP-mannose (GDP-M) was significantly enriched in basal-like tumors with HRD. GDP-M promoted cisplatin-induced DNA double-strand breaks by inducing HRD. Mechanistically, the low expression of the upstream enzyme GMPPA led to the endogenous upregulation of GDP-M, which further promoted the ubiquitin-mediated degradation of BRCA2 to inhibit HR. GMPPA expression and GDP-M could serve as predictive biomarkers for HRD and the response to PARP inhibitors. Therapeutically, we validated that the combination of GDP-M and PARP inhibitors synergistically inhibit tumor growth in multiple preclinical models. Moreover, GDP-M supplementation plus PARP inhibition activated STING-dependent antitumor immunity and further augmented the efficacy of anti-PD-1 antibodies. Conclusions: GDP-M promotes the degradation of BRCA2 and thus enhances the sensitivity of PARP inhibitors in TNBC. The combination of GDP-M, PARP inhibitors and anti-PD-1 immunotherapy may be a potential treatment strategy for HRD-low TNBC. Citation Format: Yi Xiao, Jiahan Ding, Yi-Zhou Jiang, Zhi-Ming Shao, Genhong Di, Fan Yang, Xiaoqing Song. GDP-mannose enhances the efficacy of PARP inhibitors and immunotherapy in triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-16-03.

Abstract PO2-25-02: The microbial metabolite trimethylamine N-oxide promotes antitumor immunity in triple-negative breast cancer

Abstract Immunotherapy has achieved limited success in patients with triple-negative breast cancer (TNBC), an aggressive disease with a poor prognosis. Commensal microbiota have been proven to colonize the mammary gland, but whether and how they modulate the tumor microenvironment remains elusive. We performed a multiomics analysis of a cohort of patients with TNBC (n = 360) and found genera under Clostridiales, and the related metabolite trimethylamine N-oxide (TMAO) was more abundant in tumors with an activated immune microenvironment. Patients with higher plasma TMAO achieved better responses to immunotherapy. Mechanistically, TMAO induced pyroptosis in tumor cells by activating the endoplasmic reticulum stress kinase PERK and thus enhanced CD8+ T cell-mediated antitumor immunity in TNBC in vivo. Collectively, our findings offer new insights into microbiota-metabolite-immune crosstalk and indicate that microbial metabolites, such as TMAO or its precursor choline, may represent a novel therapeutic strategy to promote the efficacy of immunotherapy in TNBC. Citation Format: Hai Wang. The microbial metabolite trimethylamine N-oxide promotes antitumor immunity in triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-25-02.

Abstract PO2-15-10: Identifying new immune-related biomarkers in TNBC with a look at PD-L1 cell-autonomous role

Abstract Background The programmed death ligand 1 (PD-L1) has recently emerged as a target immunotherapy in Triple-negative breast cancer (TNBC). However, the tumor-intrinsic role of PD-L1 and its pathway still needs to be fully clarified. Recently, a close association between CD73, PD-L1, cancer cell steaminess and epithelial-to-mesenchymal transition phenotypes is emerging. In addition, we have previously demonstrated that miR-320a and miR-145 target PD-L1, whereas miR-30 family is known to target CD73. In this study, we aimed at investigating the role of these miRNAs as prognostic and predictive dynamic biomarkers for immunotherapy in TNBC. Additionally, we investigated whether PD-L1 exerts cellular autonomic functions in TNBC beyond its role in the immune checkpoint. Results CD73 and PD-L1 expression was assessed in a panel of breast cancer lines and the non-malignant breast cells MCF-10. We showed a significantly higher CD73 and PD-L1 expression in MDA-MB-231 (TNBC, Basal B) than in MCF-10. Conversely, in MCF-7 cells (HR +, Luminal A), CD73 and PD-L1 expression is lower than in MCF-10. Furthermore, we showed that low PDL1 expression is correlated with high levels of miR-320a and miR-145, and CD73 expression is inversely related to miR-30 expression. These putative biomarkers were also validated in two patients with different PD-L1 status. From the analysis of the data obtained by real-time qRT-PCR, we found high plasma level of CD73 and low plasma level of miR-320a, miR-145 and miR-30 in the patient with PD-L1 positive BC. On contrary, in the patient with PD-L1-negative BC, we found low plasma level of CD73 and high plasma level of miR-320a, miR-145 and miR-30. Furthermore, to explore whether PD-L1 could also have an intrinsic role in tumor development and invasiveness, we used stably MDA–MB-231 PD-L1-silenced cells. We found that cell growth, colony formation, migration rate and the ability to form spheres are consistently reduced upon shRNA-mediated PDL1 silencing. In particular, we observed a reduction in the number, diameter and volume of the 3D spheres compared to the control cells. To further demonstrate the role of PD-L1, we treated the PDL1-high expression cells MDA-MB-231, PD-L1 silenced MDA-MB-231 clones, and PD-L1 low expression cells MCF-7 with Durvalumab, an anti-PD-L1. Interestingly, we observed that in PD-L1 silenced clones and MCF-7 cells, the sphere-forming ability was increased in presence of Durvalumab treatment. On the contrary, in MDA-MB-231, Durvalumab inhibited the sphere-forming ability. Conclusion Our data confirm that PD-L1 and CD73 are targets of miR-320a/miR-145 and miR-30 respectively. These could be potential predictive dynamic biomarkers for chemotherapy, ICIs and anti-CD73 therapeutic approach. Future validations of these biomarkers in an extensive series of TNBC patients are needed to support their use in clinical practice. Here, we further characterized the cellular autonomic role of PD-L1 in breast cancer and showed a differential role of basal PD-L1 expression in PD-L1 checkpoint inhibitors treatment efficacy. This suggests a potential role in monitoring PD-L1 expression indirect biomarkers (i.e. miR-320a, miR-145 and CD73) during ICIs treatment.

Abstract PO3-06-07: Efficacy and safety of sintilimab in combination with anlotinib plus metronomic chemotherapy in advanced triple negative breast cancer (SPACE): preliminary results of a single-arm, multicenter phase II trial

Abstract Background: Antiangiogenic drugs have demonstrated synergistic effect with anti-PD-1 antibody in advanced triple negative breast cancer (TNBC). Anlotinib is an oral multi-target tyrosine kinase inhibitor (TKI) that strongly inhibits VEGFR, PDGFR, FGFR, and c-kit. Preclinical studies showed that metronomic chemotherapy inhibited angiogenesis and enhanced the efficacy of immunotherapy in TNBC via modulation of the tumor immune microenvironment. We hereby conducted a single-arm, multicenter, phase II trial to investigate the efficacy and safety of sintilimab (anti-PD-1 antibody) plus anlotinib and metronomic chemotherapy as a potential novel therapeutic strategy in advanced TNBC and explore potential biomarkers. Methods: Forty-four cases were planning to be included in this trial. The eligible patients who had received no more than two lines of chemotherapy for metastatic disease were enrolled and received sintilimab (200 mg iv q3w) and anlotinib (12 mg po d1-14 q3w) plus capecitabine (500 mg po, tid) or vinorelbine (40 mg po, tiw) until disease progression or intolerable toxicity. The primary endpoint is objective response rate (ORR) and secondary endpoints are disease control rate (DCR), progression free survival (PFS), and overall survival (OS). The safety profile has also been assessed. Results: As of April 2023, a total of 44 patients were enrolled, and 42 patients were evaluable for efficacy. 3 patients (7.1%) achieved complete response (CR). 6 patients (14.3%) achieved partial response (PR). 25 patients (59.5%) achieved stable disease (SD).The ORR is 21.4% (95%CI 0.103-0.368) and DCR is 81.0% (95%CI 0.810-0.659). The median PFS was 5.06 months (95%CI 2.051-8.069). The most common grade 1 or 2 adverse events (AEs) include elevated thyroid stimulating hormone (52.38%, 22/42), elevated bilirubin (23.81%, 10/42), hand-foot syndrome (22.22%, 8/42), leukopenia (16.67%,7/42), nausea (14.29%, 6/42). Grade 3 AEs include elevated bilirubin (2.38%, 1/42), hypertension (2.38%, 1/42) and herpes zoster (2.38%, 1/42). No grade 4 or 5 AEs occurred. Conclusions: Our date showed that sintilimab in combination with anlotinib plus metronomic chemotherapy have shown favorable efficacy and acceptable safety profile in patients with advanced TNBC. Clinical trial information: ChiCTR2100044725 Citation Format: huihui Li, Dongdong Zhou, Zeshun Yv, Yuqian Liao, Jie Huang, Shujuan Sun, fangchao zheng, Baojiang Li, Shu Fang, Ling Qiang, Guohua Ren, Bing Bu, Pengfei Qiu, Xinzhao Wang, Chao Li, Fangli Cao, Qian Shao, Dali Han, Lihua Song, Baoxuan Zhang, Bingjie Fan, Liang Xu, Xuemei Xie, Xianguang Zhao, Lanping Liu, Wanlong Li, Zhenbo Wang, Changmin Liu, Hui Fu, Xiao Sun, Zhiqiang Shi, Fengxiang Li. Efficacy and safety of sintilimab in combination with anlotinib plus metronomic chemotherapy in advanced triple negative breast cancer (SPACE): preliminary results of a single-arm, multicenter phase II trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-06-07.

Abstract PO4-19-10: Selective Avoidance of Sentinel Lymph Node Biopsy After Neoadjuvant Chemotherapy In HER-2 Positive/Triple Negative Breast Cancer Patients With Excellent Radiologic Response to the Breast and Axilla

Abstract Background Neoadjuvant chemotherapy (NACT) for breast cancer (BC) is downstaging inoperable tumors into operable tumors and de-escalating breast/axillary surgery. According to advanced NACT, dual human epidermal growth factor-2 (HER2) blockage in HER2-positive BC, carboplatin, and immunotherapy in triple-negative breast cancer (TNBC) revealed pathologic complete response (ypCR) rates of up to 68% and 80%, the indications for NACT have expanded to early BC and the expected ypCR rate has increased. Therefore, it may be reasonable to consider omitting surgery in cases with excellent responses to NACT. Although sentinel lymph node biopsy (SLNB) is minimally invasive, some patients still experience complications such as lymphedema, pain, sensory loss, and axillary web syndrome. Several recent retrospective studies and pilot prospective studies have reported axillary pathologic complete response (ApCR) is highly correlated with breast pathologic complete response (BpCR). In cN0 with HER2+ BC and TNBC, the ApCR rate in BpCR after NACT was reported up to 100%, and in cN1 with HER2+ BC and TNBC, the ApCR rate in BpCR after NACT was reported about 85%. There are many studies predicting BpCR using radiologic imaging such as breast mammography (MMG), ultrasonography (USG), and magnetic resonance imaging (MRI). However, the combination of tri-modality (MMG, USG, MRI) could not confirm BpCR. The current study is screened in cT1-3N0-1M0, HER2+ or TNBC, and planned breast-conserving surgery (BCS) patients after NACT with excellent response at the combination of tri-modality imaging (breast MMG, breast USG, breast MRI) and with expected BpCR at physical examination. Methods Trial design The ASLAN trial is a prospective, multicenter, single-arm, clinical study. The five tertiary care hospitals in South Korea are participating which are members of the Korean Breast Cancer Study Group (KBCSG). Eligibility Inclusion criteria: women aged 20-69; cT1-3N0-1M0; HER2+ or TNBC (defined by ER-negative (< 10% positive cells in IHC) and PR-negative (< 10% positive cells in IHC)); expected complete remission at physical examination and radiological expected Tumor size ≤ 2cm or non-mass enhancement ≤ 4cm at breast MRI after standard NACT; planned breast-conserving surgery (BCS) with whole-breast irradiation; ECOG performance status 0-1. Exclusion criteria: SLNB before NACT; previous axillary surgery; bilateral BC; pregnancy. Intervention In eligible patients, BCS was performed. After BCS, patients who showed BpCR are enrolled with the omission of SLNB. Patients with no BpCR proceed with routine axillary surgery. Sample size calculation The assumption for an acceptable 5-year RFS ≥84% is based on previous study findings. The calculated total case number for per-protocol analysis is N=178. By Jun 2023, 153 patients had been enrolled and the target accrual of 178 patients is expected to be complete by Dec 2023. Study outcomes Primary endpoint: 5-year recurrence-free survival. Secondary endpoint: local recurrence-free survival, breast cancer-specific survival, overall survival, ipsilateral axillary recurrence interval, distant metastasis-free survival, contralateral breast-free survival, re-operation rate according to breast biopsy after NACT, adverse event, and quality of life. Accrual Plans The first patient was enrolled on Sep 2021. Among 223 patients who were screened, 153 patients have been enrolled in Jun 2023. We plan to complete the target accrual by Dec 2023. Conclusion The primary aim of the ASLAN trial is to demonstrate the oncologic safety of omitting axillary surgery for the excellent response after NACT in HER2+ or TNBC, early breast cancer patients undergoing BCS and adjuvant RT ASLAN trial, altogether with the EUBREAST-01 and ASCIS trials, will answer the very important question that may alter the axillary surgery in highly selected patients after NACT Citation Format: Jai Min Ryu, Wonshik Han, Han-Byoel Lee, Sung Gwe Ahn, Hee Jeong Kim, Hyung Seok Park, Ji Soo Choi, Hyunjun Lee, Hae Young Kim, Jeong Eon Lee. Selective Avoidance of Sentinel Lymph Node Biopsy After Neoadjuvant Chemotherapy In HER-2 Positive/Triple Negative Breast Cancer Patients With Excellent Radiologic Response to the Breast and Axilla [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-19-10.

Identification of immune-related prognostic biomarkers in triple-negative breast cancer.

Triple-negative breast cancer (TNBC), a type of breast cancer, lacks immune-related markers that can be used for prognosis or prediction. Therefore, we created a predictive framework for TNBC using a risk assessment. Our previous study group consisted of 360 individuals who were diagnosed with TNBC through pathology using RNA sequencing and had clinical data from Fudan University Shanghai Cancer Center (FUSCC). A risk scoring model was constructed using the Cox regression method with the least absolute shrinkage and selection operator (LASSO). A multivariate Cox regression analysis was utilized to develop the prediction model, which was then assessed using the consistency index and calibration plots. The validation cohort of The Cancer Genome Atlas (TCGA) TNBC confirmed the strength of the signatures' predictive value. The prognostic risk score model included 12 genes: TDO2, CHIT1, CARML2, HLA-C, ADIRF, C19orf33, CA8, AHNAK2, RHOV, OPLAH, THEM6, and NEBL. The receiver operator characteristic (ROC) curves for survivability values at 1, 3, and 5 years in the FUSCC TNBC cohort demonstrated area under the curve (AUC) values of 0.78, 0.83, and 0.75, respectively. These results indicated a high level of accuracy in predicting outcomes, which was further confirmed through validation using TCGA database. The patients in the high-risk group showed worse prognoses and lower levels of immune cell infiltration, specifically CD8+ T cells, than those in the low-risk group. Furthermore, the low-risk group exhibited a significant upregulation of genes that encode immune checkpoints, including CD274 and CTLA4, suggesting that immunotherapy may yield enhanced efficacy within this particular group. In conclusion, the prognostic signature consisting of 12 genes can assist in the choice of immunotherapy for TNBC.

Abstract 1145: Comparative spatial transcriptomic analysis of tumor microenvironment responses to conventional chemotherapy and immunotherapy in triple-negative breast cancer

Abstract Background: In triple negative breast cancer (TNBC), pembrolizumab in combination with cytotoxic chemotherapy has proven its efficacy and improved survival outcome. Previous studies demonstrated that PD-L1 expression, tumor mutational burden as well as certain neo-antigens were associated with response to pembrolizumab but there were no biomarkers predicting the response of pembrolizumab of TNBC in neoadjuvant setting. Therefore, tumor microenvironment (TME) evaluation would be warranted to understand the mechanism of action of pembrolizumab in TNBC. Methods: We employed the Xenium In Situ platform, a cutting-edge spatial transcriptomics tool, capable of mapping hundreds of transcripts within individual cells. Using a 380-plex gene panel, we analyzed formalin-fixed, paraffin-embedded TNBC sections treated with weekly paclitaxel with carboplatin followed by doxorubicin with cyclophosphamide (CTx) with/without pembrolizumab in neoadjuvant setting. We analyzed detailed cellular compositions of tumors, encompassing immune and stromal cells, across over one million cells and we attempted to identify various cell types and states, previously characterized through single-cell RNA sequencing, and assess their spatial heterogeneity within the TME with regard to treatment strategy and the response to treatment. Results: We collected 19 serial samples from 13 patients including 12 pre-treatment and seven post-treatment samples. Among seven post-treatment samples, one was collected after three weeks of the first treatment and one was after disease relapse. Of 13 patients, seven patients had received pembrolizumab with cytotoxic chemotherapy and four out of seven have achieved pathologic complete response (pCR). In six patients treated with only cytotoxic chemotherapy, only two achieved pCR. Our analysis revealed significant alterations in the spatial organization of cells in both the pembrolizumab with CTx and CTx-only groups. Notably, an increase in CD8 T cells and plasma cells following treatments was pronounced in post-treatment samples from patients in the pembrolizumab with CTx group. We also observed treatment-specific variations in ligand-receptor interactions between immune cells and cancer cells. Conclusion: This study provides an in-depth analysis of the TME in TNBCs in response to pembrolizumab with CTx. Utilizing the Xenium In Situ platform, we uncovered significant disparities in the immune cell population according to the application of pembrolizumab. Our findings underscore the complexity of TME responses and highlight the potential of spatial transcriptomics in informing tailored treatment strategies for TNBC. Citation Format: Do-Eon Gu, Jiyeon Hyeon, Eun Seop Seo, Hae Hyun Jung, Jonghan Yu, Seok Won Kim, Young-Hyuck Im, Ji-Yeon Kim, Woong-Yang Park. Comparative spatial transcriptomic analysis of tumor microenvironment responses to conventional chemotherapy and immunotherapy in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1145.

Abstract 3276: The effects of extracellular ATP signaling on the anticancer activities of macrophages

Abstract Among cancers in women worldwide, breast cancer has the highest incidence and the highest mortality. Patients with triple negative breast cancer (TNBC) have a markedly worse outcome in comparison to patients diagnosed with hormone-dependent and human epidermal growth factor receptor-positive breast cancers due to the aggressive and rapidly progressive nature of the disease and the deficit in specifically targeted therapies available. Therefore, there is a need for new therapeutic approaches. Extracellular ATP has been shown to cause cell death in an autocrine and paracrine manner and is also a known danger signal that causes immune activation. Our published data reveals that at high micromolar concentrations extracellular ATP (eATP) can induce cell death through the eATP-gated ion channels, purinergic P2RX7 and P2RX4 receptors. We also showed that in the context of chemotherapy treatment, eATP modulates TNBC cell death at submicromolar concentrations. A feature of the tumor microenvironment is a pronounced increase in the concentrations of eATP (micromolar) to levels that are closer to the threshold for cytotoxicity than in normal tissues (nanomolar). In addition, we discovered that chemotherapy induces the release of eATP from TNBC cells, further augmenting its concentration. Despite this, eATP is rapidly degraded to adenosine by extracellular ATPases (eATPases). It should be noted that extracellular ATP release and activation of its receptor, P2XR7, are prerequisites for the activation of a complex of proteins known as the NLRP3 inflammasome that is critical for IL-1β and IL-18 secretion and the induction of a highly inflammatory form of programmed cell death known as pyroptosis. The objective of this study was to evaluate if augmenting eATP levels using extracellular ATPase inhibitors will increase the efficacy of chemotherapy against THP-1 and differentiated THP-1 cells to undergo an inflammatory form of cell death known as pyroptosis. We treated THP-1 and differentiated THP-1 cells with increasing concentrations of the chemotherapeutic agent paclitaxel in the presence of eATPase inhibitors POM-1 and PSB 069 with eATP content and cell viability being assessed. Additionally, we examined NLRP3 inflammasome activation by immunoblot analysis. We analyzed the oligomerization of NLRP3 and ASC, the cleavage of caspase 1 and gasdermin and the secretion of IL-1β and IL-18, all components of pyroptosis. These results reveal that eATP modulates the chemotherapeutic response in the monocytic THP-1 and differentiated THP-1 cells, which could be exploited to augment the anticancer effects of macrophages on TNBC cells. These preclinical experiments could be applied to develop effective therapies that increase the depth and length of responses to chemotherapy and immunotherapy in TNBCs by augmenting pyroptosis of tumor associate macrophages through enhanced eATP. Citation Format: Jasmine M. Manouchehri, Lynn M. Marcho, Mathew A. Cherian. The effects of extracellular ATP signaling on the anticancer activities of macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3276.

A novel immune-related long noncoding RNA (lncRNA) pair model to predict the prognosis of triple-negative breast cancer.

Breast cancer (BC) is the most prevalent cancer type and is the principal cause of cancer-related death in women. Anti-programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) immunotherapy has shown promising effects in metastatic triple-negative breast cancer (TNBC), but the potential factors affecting its efficacy have not been elucidated. Immune-related long noncoding RNAs (irlncRNAs) have been reported to be involved in immune escape to influence the carcinogenic process through the PD-1/PD-L1 signaling pathway. Therefore, exploring the potential regulatory mechanism of irlncRNAs in PD-1/PD-L1 immunotherapy in TNBC is of great importance. We retrieved transcriptome profiling data from The Cancer Genome Atlas (TCGA) and identified differentially expressed irlncRNA (DEirlncRNA) pairs. Least absolute shrinkage and selection operator (LASSO) regression analysis was performed to construct a risk assessment model. Receiver operating characteristic (ROC) curve analysis indicated that the risk model may serve as a potential prediction tool in TNBC patients. Clinical stage and risk score were proved to be independent prognostic predictors by univariate and multivariate Cox regression analyses. Subsequently, we investigated the correlation between the risk model and tumor-infiltrating immune cells and immune checkpoints. Finally, we identified USP30-AS1 through the StarBase and Multi Experiment Matrix (MEM) databases, predicted the potential target genes of USP30-AS1, and then discovered that these target genes were closely associated with immune responses. Our study constructed a risk assessment model by irlncRNA pairs regardless of expression levels, which contributed to predicting the efficacy of immunotherapy in TNBC. Furthermore, the lncRNA USP30-AS1 in the model was positively correlated with the expression of PD-L1 and provided a potential therapeutic target for TNBC.

Advances in Monoclonal Antibody Therapies for Triple-Negative Breast Cancer: Immunotherapeutic and Targeted Strategies.

Triple-negative breast cancer (TNBC) presents considerable obstacles because of its highly aggressive characteristics and limited availability of specific therapeutic interventions. The utilization of monoclonal antibody (mAb)-based immunotherapy is a viable approach to tackle these difficulties. This review aims to examine the present state of mAb-based immunotherapy in TNBC, focusing on the underlying mechanisms of action, clinical applications, and existing challenges. The effectiveness of mAbs in reducing tumor development, regulating immune responses, and changing the tumor microenvironment has been demonstrated in many clinical investigations. The challenges encompass several aspects such as the discovery of biomarkers, understanding resistance mechanisms, managing toxicity, considering costs, and ensuring accessibility. The future is poised to bring forth significant advancements in the field of biomedicine, particularly in the areas of new mAbs, personalized medicine, and precision immunotherapy. In conclusion, mAb-based immunotherapy has promise in revolutionizing the treatment of TNBC, hence providing a possible avenue for enhanced patient outcomes and quality of life.

Nanoparticle-integrated dissolving microneedles for the co-delivery of R848/aPD-1 to synergistically reverse the immunosuppressive microenvironment of triple-negative breast cancer

Nowadays, effective immunotherapy against triple-negative breast cancer (TNBC) remains challenging due to the immunosuppressive tumor microenvironment. Immune checkpoint inhibitor is mostly employed to restore the activity of tumor-specific immune cells, which however brings little therapeutic outcome owing to the limited number of tumor-infiltrating CD8+ T cells and the inefficient delivery of immune drugs to the tumor tissue. Aiming to solve these problems, we herein constructed a tailor-made dissolving microneedle co-encapsulating the TLR7/8 agonist R848 and the immune checkpoint inhibitor aPD-1, termed αNP-RNP@DMN, and fabricated it as a transdermal drug delivery system. This well-designed microneedle patch, endowed with efficient tumor drug delivery ability, was able to mature tumor-infiltrating dendritic cells (TIDCs) and further promote the infiltration of CD8+ T cells into the tumor tissue with the aid of R848. Moreover, the introduction of aPD-1 blocked the programmed cell death protein 1/programmed cell death ligand 1(PD-1/PD-L1) immune checkpoints, synergistically reversing the immunosuppressive microenvironment of TNBC. In vivo therapeutic results demonstrated that αNP-RNP@DMN not only significantly prolonged the survival time of 4T1 tumor-bearing mice, but also inhibited tumor recurrence and lung metastasis after surgery, implying the great potential of this effective drug delivery system for enhanced immunotherapy of superficial tumors. Statement of significanceThe limited number of tumor-infiltrating CD8+ T cells and the inefficient delivery of immune drugs to the tumor tissue hinder the effective immunotherapy of triple-negative breast cancer (TNBC). Herein, a dissolving microneedle co-encapsulating TLR7/8 agonist R848 and immune checkpoint inhibitor aPD-1 was developed and fabricated as a transdermal drug delivery system. This tailor-made microneedle patch not only promoted drug accumulation in tumor sites in a safe and painless manner, but also lifted the immune-suppressive state of tumor-infiltrating dendritic cells (TIDCs). The activated TIDCs further enhanced T-cell infiltration into the tumor tissue, thus successfully boosting the therapeutic efficacy of aPD-1. This study demonstrated that this well-designed microneedle patch could be served as an effective drug delivery system for enhanced immunotherapy of TNBC.

Single-cell RNA-seq reveals the metabolic status of immune cells response to immunotherapy in triple-negative breast cancer

Immune checkpoint blockade (ICB) therapy offers promise in the treatment of triple-negative breast cancer (TNBC); however, its limited efficacy in certain TNBC patients poses a challenge. In this study, we elucidated the metabolic mechanism at ‘sub-subtype’ resolution underlying the non-response to ICB therapy in TNBC. Here, an analytic pipeline was developed to reveal the metabolic heterogeneity, which is correlated with the ICB outcomes, within each immune cell subtype. First, we identified metabolic ‘sub-subtypes’ within certain cell subtypes, predominantly T cell subsets, which are enriched in ICB non-responders and named as non-responder-enriched (NR-E) clusters. Notably, most of NR-E T metabolic cells exhibit globally higher metabolic activities compared to other cells within the same individual subtype. Further, we investigated the extra-cellular signals that trigger the metabolic status of NR-E T cells. In detail, the prediction of cell-to-cell communication indicated that NR-E T cells are regulated by plasmatic dendritic cells (pDCs) through TNFSF9, as well as by macrophages expressing SIGLEC9. In addition, we also validate the communication between TNFSF9+ pDCs and NR-E T cells utilizing deconvolution of spatial transcriptomics analysis. In summary, our research identified specific metabolic ‘sub-subtypes’ associated with ICB non-response and uncovered the mechanisms of their regulation in TNBC. And the proposed analytical pipeline can be used to examine metabolic heterogeneity within cell types that correlate with diverse phenotypes.

Light-inducible nanodrug-mediated photodynamic and anti-apoptotic synergy for enhanced immunotherapy in triple-negative breast cancer.

Triple negative breast cancer (TNBC) exhibits limited responsiveness to immunotherapy owing to its immunosuppressive tumor microenvironment (TME). Here, a reactive oxygen species (ROS)-labile nanodrug encapsulating the photosensitizer Ce6 and Bcl-2 inhibitor ABT-737 was developed to provoke a robust immune response via the synergistic effect of photodynamic therapy (PDT) and the reversal of apoptosis resistance. Upon exposure to first-wave near-infrared laser irradiation, the generated ROS triggers PEG cleavage, facilitating the accumulation of the nanodrug at tumor region and endocytosis by tumor cells. Further irradiation leads to the substantial generation of cytotoxic ROS, initiating an immunogenic cell death (ICD) cascade, which prompts the maturation of dendritic cells (DCs) as well as the infiltration of T cells into the tumor site. Meanwhile, Bcl-2 inhibition counteracts apoptosis resistance, thereby amplifying PDT-induced ICD and bolstering antitumor immunity. As a result, the ROS-sensitive nanodrug demonstrates a potent inhibitory effect on tumor growth.

The use of MFAP2 for diagnosis, prognosis and immunotherapy of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is characterized by significant heterogeneity, presenting a formidable challenge with a poor prognosis and a deficiency of efficacious treatment options. In this comprehensive study, we investigated the multifaceted role of Microfibril-associated glycoprotein 2 (MFAP2) in TNBC using a combination of bioinformatics analysis involving Gene Expression Omnibus (GEO), OncoDB, UALCAN, Human Protein Atlas (HPA), TIMER, STRING, DAVID, and GSCA databases and in vitro experiments, such as cell culture, MFAP2 gene knockdown, RT-qPCR, western Blot, colony formation, Cell counting kit-8, and wound healing assays. Our findings demonstrated a significant up-regulation of MFAP2 mRNA in TNBC cell lines, emphasizing its potential as a diagnostic biomarker. Validation across multiple datasets further affirmed the elevated expression of MFAP2 in TNBC tissues, underscoring its prognostic relevance. Notably, our study revealed a correlation between MFAP2 expression and immune cell infiltration, suggesting its role in shaping the tumor microenvironment. STRING analysis unveiled interactions with proteins involved in elastic fibers and extracellular matrix constituents. Furthermore, KEGG pathway analysis highlighted enrichment in the TGF-beta signaling pathway, implicating MFAP2 in key cancer-related processes. Drug sensitivity analysis identified potential therapeutic targets, supporting MFAP2's utility in personalized treatment strategies. In vitro experiments corroborated the oncogenic impact of MFAP2, demonstrating its influence on TNBC cell proliferation and migration. These comprehensive findings position MFAP2 as a promising biomarker and therapeutic target in TNBC, offering valuable insight for future research and clinical application.