Abstract
Introduction: Circulating tumor cells (CTCs) mediate metastases, which account for 90% of solid tumor-related mortality. Compared to single cells, clustered CTCs mediate metastasis at a ...20-100 times higher efficiency and are associated with lower overall survival in breast cancer (BC). We have recently identified a new mechanism of CTC cluster formation through cellular aggregation instead of cohesive shedding, and demonstrated that CTC clusters have enhanced stemness (Cancer Discovery, 2019). However, the cellular heterogeneity and molecular mechanisms underlying CTC cluster formation and polyclonal metastasis have yet to be fully elucidated. We hypothesize that molecular drivers of metastasis initiation enhance cancer stemness and CTC cluster formation, and serve as a novel therapeutic target for BC metastasis.
Methods: Using single cell RNA sequencing, we compared tumor cells from the primary breast tumor site and lung metastases of BC patient-derived xenografts. We identified genes specifically expressed in the lung metastases and determined their functional importance in CTC clustering, cancer stemness, and lung colonization. We performed proteomic and transcriptomic analyses as well as machine learning to elucidate the downstream signaling pathways and protein structural basis involved in CTC cluster formation and lung metastasis. Finally, we explored therapeutic intervention options in blocking CTC cluster formation and lung metastasis.
New results: Compared to the primary breast tumor cells, we identified a stemness gene signature enriched in a subpopulation of the CD44+ lung metastases, with 30-60 fold higher expression of CD34, CD36, ICAM1, VCAM1, ZEB1, ALDH1A1, TGFBR2 and TSPAN8. Analysis of patient blood samples (N=40) revealed that CD44 and many of these new candidate proteins were enriched in CTC clusters in comparison to single CTCs. We then examined the CSC-related properties of these tumor cells, such as tumorigenesis, sphere formation, and lung metastasis. Knockdown of selected surface molecules (e.g. ICAM1) significantly reduced the efficiency of lung metastasis of BC cells in vivo. A subset of ICAM1+/CD44+ tumor cells had increased stemness and tumor growth upon orthotopic implantation in vivo. Knockdown of ICAM1 dramatically reduced the self-renewal ability in mammosphere formation of breast tumor cells in vitro. In addition, our studies also revealed that these tumor cells cluster through CD44 and other surface protein-mediated homophilic binding between two neighboring tumor cells. Neutralizing antibodies significantly blocked tumor cluster formation and lung colonization.
Conclusions: We identified new molecular mediators of CTC aggregation and lung metastasis in BC. We anticipate that specific blockade of tumor clustering could decrease cancer progression and improve survival of BC patients.
Citation Format: Rokana Taftaf, Xia Liu, Salendra Singh, Yuzhi Jia, David Scholten, Youbin Zhang, Andrew Davis, Carolina Reduzzi, Yue Cao, Yang Shen, Massimo Cristofanilli, William A. Muller, Vinay Varadan, Huiping Liu. Single cell RNA sequencing-based identification of molecular drivers in circulating tumor cell cluster formation and lung metastasis abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2603.
Abstract
Introduction: Novel high throughput genomic technologies are enhancing the ability to dynamically characterize cancer on a granular level. Although Next Generation Sequencing (NGS) is ...becoming part of the common practice, little is known on the role of a characterization on a gene variant level. Large heterochromatic regions or blocks are a characteristic feature in the autosomes 1, 9, 16 and the blocks contain about 10% of the human genome. The heterochromatic regions consist of highly repetitive DNA of the classes Sat I to IV. Thus, these specific regions are preferential positions for genome instability. The aim of this study was to explore the feasibility of a gene variant-oriented characterization for variants interplay discovery and to explore the role of chromosome stability in gene variants incidence. Methods: This study analyzed a pilot cohort of 35 metastatic breast cancer (MBC) patients (pts) treated and characterized for CTCs and circulating tumor DNA (ctDNA) at Northwestern University (Chicago, IL). ctDNA was analyzed using the PredicinePLUS™ NGS 180-gene panel (Predicine Inc, CA). Associations between gene variants and clinico-molecular characteristics were tested through Fisher’s exact test. Chromosomes 1, 9, 16 where defined as instable (instable_chr) based on the presence of highly repeated sequences. Results: An overall set of 35 samples was analyzed, and the main variants were detected in the ARID1A (40%), ATM (20%), DNMT3A (37%), ESR1 (20%), PIK3CA (26%), and TP53 (49%) genes. A total of 448 gene variants were detected through the NGS panel and across them, ARID1A accounted for 5.13%, ATM 2.23%, DNMT3A 4.91%, ESR1 4.02%, PIK3CA 2.23%, and TP53 for 6.03%. Among the genes mutated, the DNMT3A:c.2644C>T, DNMT3A:c.2645G>A, ESR1:c.1138G>C,ESR1:c.1609T>A, ESR1:c.1610A>C, ESR1:c.1613A>G, PIK3CA:c.1624G>A were the most detected. Thirty-eight copy number variations (CNV) and 3 splicing variants were observed. Notably, 55.6% of detected variants were found in instable_chr. No significant differences were observed between instable_chr variants and MBC subtype. Interestingly, ARID1Aaberrations were significantly linked to ATM, ESR1 (P=0.006) and PIK3CA with ESR1 (P= 0.019). Consistently with literature, ATM and TP53 variants were mutually exclusive (P<0.0001). Intriguingly, both ATM and TP53 were associated with a higher incidence of variants affecting genes in instable_chr. Conclusions: MBC is often described as a non-gene-addicted disease, rendering the onset of gene variants a multi-factor phenomenon. The present results suggest a role for both chromosomal intrinsic instability and DNA repair impairment in this process, with a potential down-stream selection mediated by treatment resistance. Future studies are warranted to further validate this proof of concept approach.
Citation Format: Lorenzo Gerratana, Qiang Zhang, Ami N Shah, Alessandra Franzoni, Jianjun Yu, Shidong Jia, Andrew A Davis, Youbin Zhang, Firas Wehbe, Amir Behdad, Leonidas C Platanias, William J Gradishar, Massimo Cristofanilli. Next generation sequencing-based gene variant-oriented characterization in metastatic breast cancer: An innovative analysis using ctDNA abstract. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-10.
Abstract
Background: Breast cancer (BC) is a heterogeneous disease comprising different clinical, histopathological, and molecular subtypes. Hormone receptor-positive (HR+) advanced BC cancer, in ...particular the inflammatory BC (IBC) represents a significant clinical challenge because of the development of endocrine resistance during either adjuvant or metastatic treatment translating to poor outcome. We aim to develop unique PDX tumor models and 3D spheroid/organoid cultures that recapture advanced IBC using pleural effusions or/and circulating tumor cells (CTCs) as tumor resource and that can help understand the mechanism of IBC metastasis for new drug development. Methods: Five samples of pleural effusion-derived tumor cells or circulating tumor cells (CTCs) from three advanced IBC patients were obtained from Northwestern Memorial Hospital to establish IBC PDX tumor model in immunodeficient NSG female mice via subcutaneous or breast fat pad xenografting and develop the derived 3D organoid/spheroid cultures for anti-cancer drug evaluation. STR profiling against with original patient tumor DNA was conducted to confirm the tumor authentication. Results: Three developed IBC PDX tumor models (one HR+-IBC and two triple negative (TN)-IBC) were demonstrated by pathology to have highly heterogeneous characteristics and metastatic features similar to the original patient tumor. NSG mice xenografted with original TN-IBC pleural effusion, or derived spheroid cultures with varying passage numbers (<3 passages or >6 passages) manifested different inflammatory clinical symptoms. Mice xenografted with one case of original TN-IBC pleural effusion cells (~40% of tumor-associated macrophages and ~60% of tumor cells) developed palpable tumors and was shown to have the most IBC-like characteristics including reddish skin and ulcerative lesions. They were sacrificed 2 months after xenograft due to declining health. Mice xenografted with >6 passages or <3 passages of spheroid cultures manifested little to moderate skin lesions and developed tumor sizes of ~1 cm within 2-3 months while having a longer life span. Liver and lung metastases were observed in the breast fat pad of xenografted PDX tumor mice with one case of original HR+-IBC pleural effusion. 3D tumor spheroid/organoid cultures were successfully established from original IBC pleural effusion or/and IBC PDX tumor cultures. Spheroid generated from HR+-IBC pleural effusion showed phenotypic plasticity: the HR+-IBC spheroid cells display both tumor and macrophage cell characteristics and spheroid cultures can convert to adhesive cell phenotypes with addition of condition media. These spheroid/organoid cultures are used for evaluation of new drug strategies such as selectively targeting ER degraders (SERDS), CDK4/6 inhibitor, or Tyrosine kinase inhibitors for treatment of HR+-IBC or TN-IBC. Conclusions: Our results suggested 3D spheroid cultures and PDX tumor models from advanced IBC recapture aggressive features of the disease and can be an ideal model for the mechanism study of metastatic advanced IBC and preclinical testing of novel agents in IBC and endocrine resistant disease.
Citation Format: Wenan Qiang, Zhangfeng Zhong, Pamela Monahan, Kristy Tse, Youbin Zhang, Qiang Zhang, Lorenzo Gerratana, Andrew Davis, Demirkan Gursel, Reiner Bleher, Jian-Jun Wei, Charles D. James, Thomas V. O’Halloran, Massimo Cristofanilli. Development of patient-derived xenograft tumor models and 3D spheroid culture from advanced hormone receptor-positive inflammatory breast cancer patients for evaluation of new therapeutics abstract. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-03-06.
Abstract
Background: Relapse and endocrine resistance are major clinical challenges in the management of patients with MBC because it is a dynamic phenomenon including development of ESR1 mutations. ...The monitoring of ctDNA and circulating tumor cells (CTCs) in patients with MBC may predict metastasis and prognosis. We previously reported that ctDNA can be used to evaluate tumor heterogeneity (2022 AACR-#1950 and 2022 ASCO-#1057). Here, we report that dynamic ctDNA ESR1 mutations is a key point associated with a shorter survival, which may help to elucidate prognosis in patients with MBC.
Methods: This study included 406 hormone receptors positive MBC patients who received systemic treatment under an IRB-approved clinical trial (NU16B06) at Northwestern University Robert H Lurie Comprehensive Cancer Center. Plasma samples were collected from each patient at multiple time points: before treatment (Time point 1), and then 3 months (Time point 2), 6 months (Time point 3), 9 months (Time point 4), 12 months (Time point 5) and 24 months (Time point 6) after initiation of systemic treatment respectively. Plasma ctDNA was isolated using a Qiagen circulating nucleic acid kit, and then was analyzed using the Guardant360 Health next-generation sequencing (NGS)-based assay. All statistical analyses were conducted Mann-Whitney U test by IBM SPSS version 23.0.
Results: Of the 406 patients, ESR1 mutations were found in 18 hotspots from 59 patients (ESR1Mut, 14.5%) at either time points. There were 347 patients without any mutation (ESR1WT, 85.5%). Among the 59 patients who have ESR1 mutations, 41 (69.5%) patients have ESR1 mutations at only one time point, 6 patients (10.2%) have ESR1 mutations at 2 time pints, 5 patients (8.5%) have ESR1 mutations at 3 time points, 2 patients (3.3%) have ESR1 mutations at 4 time points, and 5 patents (8.5%) have ESR1 mutations for 5 time points respectively. The median survival times for patients in ESR1WT group is 11.5 years compared to 6.4 years for patients in ESR1Mut group (P=0.0134). This result indicated that patients without ESR1 mutations at any time point have 1.81 times longer median survival time than patients who have ESR1 mutations at any time point. Furthermore, 41 patients who have ESR1 mutation at only one time point have longer survival time (7.1 years) compared to 18 patients who have ESR1 mutation at more than 2 time points (5.1 years) (P<0.01).
Conclusions: Dynamic development of ctDNA ESR1 mutations at different time points during the treatment significantly correlated with prognosis and survival of patients with metastatic breast cancer. Longitudinal dynamics of ESR1 mutation for treatment monitoring may offer important message for minimal residual disease and expand the early predictive role of prognosis for clinical decision-making in metastatic breast cancer.
Citation Format: Qiang Zhang, Jianhua Jiao, Lorenzo Gerratana, Paolo D’Amico, Seema Singhal, Youbin Zhang, Andrew A. Davis, Ami N. Shah, William Gradishar. Dynamic development of ESR1 mutations in circulating tumor DNA (ctDNA) is associated with prognosis of patients with metastatic breast cancer (MBC) abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2220.
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Background: Liquid biopsy provides real-time data about prognosis and actionable mutations in MBC. The aim of this study was to explore the combination of ctDNA analysis and CTCs ...enumeration in estimating target organs more susceptible to MBC involvement. Methods: This retrospective study analyzed 85 MBC patients (pts) characterized for both CTCs and ctDNA at baseline. CTCs were isolated through the CellSearch kit (Menarini Silicon Biosystems, PA), while ctDNA was analyzed using the Guardant360 NGS-based assay (Guardant Health, CA). Pts with ≥ 5 CTC/7.5 ml of blood were defined as Stage IV aggressive as previously reported (Cristofanilli et al 2019). Statistical associations were explored through uni- and multivariate logistic regression and Fisher’s exact test. Results: 37% of pts were diagnosed with hormone receptor positive (HRpos) MBC, 26% with HER2-positive MBC and 37% with triple negative MBC (TNBC), 28 pts (33%) were defined as stage IV aggressive. The most observed metastatic sites were bone (37%), lymph nodes (29%), lung (27%) and liver (25%). In multivariate analysis, IBC and ESR1 mutations were the only significant factors associated with liver metastases (respectively, OR 0.12, P = 0.038 and OR 24.01, P = 0.019), while no associations were found with respect to lung localizations. Intriguingly, all HRpos MBC pts with ESR1 mutations had bone metastases (P = 0.022), while IBC and Stage IV aggressive were independently associated with bone metastases (respectively OR 0.10, P = 0.006 and OR 19.92, P = 0.003). FGFR1 and NF1 were associated with lymph node localizations (OR 3.68, P = 0.046, OR 4.39, P = 0.031, respectively), while CDK6 and TP53 alterations were associated with serosal involvement (OR 14.34, P = 0.029, OR 0.08, P = 0.031, respectively). Notably, TNBC and IBC were both associated with soft tissue spreading (respectively OR 3.7, P = 0.011, OR 2.79, P = 0.018). Conclusions: These results suggest that ctDNA and CTCs enumeration could give useful insights on MBC organotropism, suggesting a possible role for future monitoring strategies that dynamically focus on high-risk organs defined by tumor biology.
Abstract
Introduction: Although CTCs display the same spatial and temporal heterogeneity as the primary tumor, they represent a privileged window to disclose mechanisms of metastases. We previously ...reported that overexpression of HER2 in CTCs is associated with worse prognosis in MBC (2018 AACR #5195). Herein, we report a new finding of heterogeneity profiling for HER2 positive CTC by using a new approach for single CTC isolation and sequencing.
Methods: Whole blood sample (7.5ml/each) was collected from stage III/IV MBC patients before therapy. CTC enumeration were performed in FDA approved CellSearch™ System by targeting the EpCAM antigen for capturing CTCs which were then stained by Anti-CK-PE, DAPI, anti-CD45-APC and anti-HER2-FITC. The single HER2+ CTCs were isolated by using DEPArrayTM System (Menarini). The single cell DNA was isolated and the initial library was prepared by SMARTer® PicoPLEX® Gold Single Cell DNA-Seq Kit, and the exome capture was performed by Twist Human Core Exome EF Multiplex Complete Kit. The sequencing was prepared by NextSeq 500 mid output V2.5 kit and was performed on the NextSeq 500 (Illumina). It was a paired end run, 75×75 bps run with dual indexing.
Results: We identified 208 CTCs by CellSearch™ system, including 114 HER2+ CTCs and 4 CTC-clusters. Single HER2+ CTC was isolated and sequenced. The sequencing data was processed following the GATK pipeline and annotated using SnpEff. There were 362,015 counts (58.23%) for intron variants, 104,011 counts (16.73%) for intergenic variants, 56,678 counts (9.19%) for exon variants, 41,133 counts (6.67%) for downstream genes, 33,903 counts (5.45%) for upstream genes and others (3.73%). There were 44 genes (64 sites) variants were identified to have highest impact effect (≥20) on HER2+ CTC chromosome, among which TP53, PIK3CA, ESR1, PIK3R1 variants matched the most recent genomic characterization report for MBC tissue (Bertucci F. Nature 2019) when the XYLB, CYP17A1, CA2 and CAD variants were firstly reported. More important, although overexpression of PTP1B (PTPN1) was implicated in the development of MBC, our evidences firstly demonstrated that PTPN1 mutation is the most important variant (≥20) in CTCs for MBC. Furthermore, we also found that ESR1 mutation in CTC correlated with ESR1 status in ctDNA. Except for the high impact variants, there were 42 genes (66 sites) and 83 genes (136 sites) have medium (10-19) and significant (5-9) impact variants respectively.
Conclusion: Genomic characterization of HER2 positive single CTCs will elucidate new specific gene alterations (e.g. PTPN1) associated with disease metastasis, which will improve understanding of this critical process in MBC and lead to the development of novel drugs aimed at their eradication using molecularly driven therapies for this deadly condition.
Citation Format: Qiang Zhang, Lorenzo Gerratana, Kara L. Pivarski, Xinkun Wang, Zhe Ji, Emily Stroup, Elena Vagia Vagia, Andrew Adam Davis A. Adam Davis, Ami Shah, Katy L. Kerby, Lisa Flaum Flaum, Firas Wehbe, Youbin Zhang, Wenan Qiang, Amir Behdad, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. Genetic heterogeneity profiling for HER2 positive single circulating tumor cell (CTC) in metastatic breast cancer (MBC) by application of DEPArrayTM System and single cell DNA sequencing for HER2 positive single circulating tumor cell (CTC) in metastatic breast cancer (MBC) by application DEPArrayTM system and single cell DNA sequencing abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3120.
Abstract
Background: Circulating tumor cells (CTCs) in breast cancer (BC) are commonly defined as epithelial cells (EPCAM and cytokeratin (CK) positive), lacking the universal blood cell marker CD45. ...Nonetheless, CTCs expressing both CK and CD45 (= dual-positive, DP cells) can be observed in the blood of cancer patients. Early evidence suggests that DP cells might derive from the fusion of tumor cells and macrophages, and we have previously demonstrated that they present aberrant genomes and are associated with worse prognosis in BC 1,2. Here, to further investigate the mechanisms/pathways underlying their presence, we analyzed the association between DP cells and circulating tumor DNA (ctDNA) alterations. Methods: Blood samples were collected from patients with advanced BC (aBC), before starting a new line of therapy. All patients were enrolled in a prospective clinical trial. For CTC and DP cells analysis, 7.5 ml of blood collected in CellSave® tubes was processed with the FDA-approved CellSearch® platform (positivity cutoffs were ≥1 cell for DPcells and ≥5 cells for CTCs). For ctDNA analysis, plasma was collected from Streck stabilizing tubes and analyzed with the Guardant360™ NGS platform for the detection of somatic single nucleotide variants (SNVs), insertions/deletions (indels), gene fusions/rearrangements and copy number variations (CNVs), which were then classified into pathways based on previously defined profiles generated on the Cancer Genome Atlas database (RTK, RAS, RAF, MEK, NRF2, ER, WNT, MYC, P53, cell cycle, notch, PI3K). Associations between ctDNA-detected gene alterations and circulating cell types were analyzed through chi square test, while mutant allele frequency (MAF) and number of detected alterations (NDA) were tested by Mann Whitney test. Results: We analyzed blood samples from 169 patients with luminal-like (n=80), HER2+ (n=34) and triple-negative (n=52) aBC. DPcells were detected in 85 patients (50.3 %, range 0-53), of which 40 (47 %) were CTC-positive and 45 (53%) CTC-negative. Somatic ctDNA alterations were detected in all analyzed samples. In the overall population, the presence of ≥1 DPcell was associated with SNVs in the cell cycle pathway (p = 0.043), a numerically higher incidence was also observed for CNVs in this pathway. SNVs and CNVs in the cell cycle pathway were associated with CTCs ≥ 5 as well (p = 0.005 and p = 0.003, respectively). Moreover, associations with CTCs ≥ 5 were observed for RTK SNVs and CNVs (p = 0.041 and p = 0.046, respectively), PI3K SNVs and CNVs (p = 0.006 and p = 0.007, respectively), MYC SNVs and CNVs (p = 0.042). No associations were observed in terms of MAF and NDA. In the luminal-like subgroup an association was highlighted for CNVs in the cell cycle pathway, p = 0.038. CTCs ≥ 5 were associated with PI3K SNVs (p = 0.031). In the triple-negative subgroup DPcells were associated with SNVs in the RAF pathway (p = 0.041), whereas CTCs ≥ 5 were associated with PI3K SNVs and CNVs (p = 0.044 and p = 0.024, respectively) and RTK SNVs (p = 0.008). In the HER2 positive subgroup, a higher MAF and number of detected SNVs was observed for samples with ≥1 DPcell (p = 0.0286 and p = 0.0099, respectively). Conclusions: The study analyzed ctDNA features associated with canonical and CK+/CD45+ CTCs, showing differential gene alteration profiles. Cell cycle pathway SNVs were common in both CTC populations, while other pathways (RTK, PI3K, MYC and RAF) were significantly altered in a mutually exclusive pattern. These results suggest that DPcells might have a different biological meaning compared to canonical CTCs. More studies need to be conducted to better characterize this understudied CTC subpopulation and understand their specific contribution to cancer progression. References: 1) Reduzzi C. et al., Semin Cancer Biol. 2020;60:344-350. DOI:10.1016/j.semcancer.2019.10.008 2) Reduzzi C. et al., Journal of Clinical Oncology 40, no. 16_suppl (June 01, 2022) 1093-1093. DOI: 10.1200/JCO.2022.40.16_suppl.1093
Citation Format: Carolina REDUZZI, Lorenzo Gerratana, Youbin Zhang, Maroua MANAI, Paolo D’Amico, Andrew A. Davis, Jeannine Donahue, Ami N. Shah, Massimo Cristofanilli. Association between CK+/CD45+ circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) alterations in advanced breast cancer patients abstract. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-06.
Abstract Background: Several prospective, randomized clinical trials showed that the CDK4/6 inhibitor palbociclib in combination with either letrozole or fulvestrant significantly improves ...progression-free survival (PFS) in patients with ER+/HER2- metastatic breast cancer (MBC). However, in some cases, there is development of endocrine resistance and ultimate disease progression. Molecular analysis performed in tissue specimens collected in the PALOMA-3 study, comparing fulvestrant and Palbociclib versus fulvestrant placebo, demonstrated that one of the genes associated with an increased clinical benefit is SIRT3 (Sirtuin 3) which is thought to control mitochondrial integrity and metabolism (Cristofanilli M. et al., Lancet Oncol. 2016). This study aimed to evaluate in preclinical models the predictive role of SIRT3 in ER+/HER2- breast tumors. Methods: Using lentiviruses we generated MCF-7 and T47D cells stably expressing either control shRNA (sh ctr) or shRNA targeting SIRT3 (shSIRT3). With these models, we studied cell viability, tumor growth, apoptosis, and autophagy in MCF-7 cells treated with fulvestrant and palbociclib as a single treatment or in combination, these either alone or in combination with shSIRT3 (knockdown). Nude mice were used for our in vivo studies, and sh ctr or shSIRT3 MCF7 cells were injected. Results: We showed in vitro that the response to palbociclib was significantly associated with levels of SIRT3 expressionas high or low in ER+ cells (p< 0.0001). Furthermore, we found that irreversible cell growth inhibition mediates the beneficial effect of palbociclib in SIRT3 high expressing cellscompared to low expression (p< 0.001).Then, we evaluated the mechanistic activity of SIRT3. We showed thatSIRT3-low expression cells induced enhanced sensitivity to palbociclib by targeting the reactive oxygen species (ROS)/autophagy axis:(i) SIRT3 regulated the mitochondrial homeostasis (e.g. glucose, lactate, pyruvate, succinates), (ii) ROS levels where lower in sh crt comparing and shSIRT3 in treated and untreated cells (p< 0.001), (iii) in terms of senescence,a higher level of positive cells for β-gal activity was found (p≤0.05) and (iv) for autophagy, our western blot analysis showed cleaved LC3 proteins. Our in vivo studies showed that SIRT3 regulated treatment response to palbociclib with a significant decrease in tumor weight and tumor volume (p< 0.01) and importantly, after immunohistochemistry analysis, we validated the role of SIRT3 in regulating the proteins involved in the autophagy/senescence balance, was mainly in epithelial cells compared to stromal cells. Conclusion: Our preclinical studies demonstrated that elevated SIRT3 expression levels sensitizes ER+/HER2- breast tumors to palbociclib treatment. This study suggested that SIRT3 expression could represent a potential predictive biomarker in HR+ MBC patients treated with Palbociclib. Citation Format: Maroua Manai, Ghada Sahraoui, Raoudha Doghri, Lorenzo Gerratana, Paolo D’Amico, Youbin Zhang, Jeannine Donahue, Ami Shah, Carolina Reduzzi, Wenan Qiang, Massimo Cristofanilli. SIRT3 as a new potential predictive biomarker for response to CDK4/6 inhibition in ER+/HER2- metastatic breast cancer patients 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 PO3-14-12.
A gene therapy-mediated approach to the delivery of antiangiogenic agents using adeno-associated virus (AAV) vectors has a number of advantages including the potential for sustained expression. The ...purpose of this study was to attempt to restrict the growth of disseminated neuroblastoma through delivery of a truncated, soluble form of the vascular endothelial growth factor receptor-2 (VEGFR-2 fetal liver kinase Flk-1), a decoy receptor for VEGF.
Mice underwent portal vein injection of vector, either AAV-CAGG-tsFlk-1 or control vector. Subsequent truncated soluble fetal liver kinase-1 (tsFlk-1) expression was confirmed and quantified by ELISA. After 8 weeks, mice were given human neuroblastoma cells through the tail vein to establish disseminated disease and were sacrificed after 40 days. The weight of liver metastasis was measured. Intrahepatic tumor microvessel density and levels of apoptosis were determined. Survival was assessed in a second cohort of mice.
After intraportal injection of AAV-CAGG-tsFlk-1, high-level, stable transgene expression was generated. Sera from these mice inhibited endothelial cell activation in vitro and Matrigel plug (BD Biosciences) neovascularization in vivo, suggesting that a systemic state of angiogenesis inhibition had been established. After neuroblastoma injection, mice expressing tsFlk-1 had a smaller tumor burden in the liver when sacrificed, as compared with control mice. The decrease in tumor weight in the liver correlated with lower intratumoral microvessel density and higher levels of tumor cell apoptosis in the tsFlk-1—treated tumors, supporting the hypothesis that decreased angiogenesis had occurred. In a second cohort of mice, survival of tsFlk-1—expressing mice after tumor cell challenge was longer than in control mice.
Longterm in vivo expression of a functional VEGF inhibitor was established using an AAV-mediated gene therapy approach, and it demonstrated antiangiogenic and anticancer efficacy in a murine model of disseminated neuroblastoma.
