Human Breast Tumors Research Articles

Mesoporous Polydopamine-Based Nanovehicles as a Versatile Drug Loading Platform to Enable Tumor-Sufficient Synergistic Therapy.

The combination of photothermal therapy and chemotherapy are developing as a promising clinical strategy but it urgently needs the high exploration of intelligent multifunctional drug delivery nanovectors. In this paper, we used a versatile method to construct mesoporous polydopamine nanovehicles (MPDA) with the dendritic mesopores loaded with a clinical chemotherapeutic drug, Doxorubicin (MPDA@DOX). The monodisperse nanoagents are spherical with a size of ∼160 nm and pore size of approximately 10 nm. MPDA could efficiently delivery DOX with π-π stacking interaction and acts as the potent photothermal agents. Importantly, MPDA@DOX are preferentially internalized by cancerous cells, then bursting drug release and local hyperthermia generation were observed in conditions representative of the cytoplasm in tumor cells that highly synergistic cell killing effect were found under 808 nm laser irradiation. The fluorescent imaging results of human breast tumor bearing murine model evidenced that MPDA delivery platform have excellent tumor precise targeting effect and in vivo tumor ablation experiment further revealed that MPDA@DOX showed markedly eradicated tumor growth capability under laser exposure. Therefore, this work provided a fascinating strategy based on biocompatible MPDA based drug delivery system for malignant tumors eradication via synergistic therapy.

Análisis de polimorfismos en los genes BRCA1 y BRCA2 en una muestra poblacional de caninos de Uruguay

In non-ovariectomized female dogs, breast tumors are the most frequent neoplasms. There are several points where canine and human breast tumors have clinical and molecular similarities. BRCA1 and BRCA2 genes have been extensively studied in both species. Regarding dogs, alterations in BRCA1 and BRCA2 have been identified in the development of breast tumors in different breeds. In this work, it was proposed to study exons 22 and 23 of the BRCA1 gene and exons 11 and 27 of the BRCA2 gene, in female dogs. It was studied two groups of female dogs, with or without mammary tumors. Regarding the genetic study of 15 loci, six were polymorphic, all of them were singles nucleotides polymorphisms (SNPs), while the other nine were monomorphic. It was obtained a low allelic variability, but at the population level, the tumor group has greater variability than the control group. On the other hand, the different analyses of possible groupings were negative, and it was not possible to clearly define groups with the parameters it was used. The foregoing may be a consequence of numerous factors such as characteristics inherent to the populations studied, such as the size of both populations; the breeds studied; tumor diversity. As it was mentioned before, the genes studied in this work have been widely related to breast cancer, both in humans and in dogs. In the former, they have been highly implicated in hereditary tumors. In dogs, it do not have that information. In the present case, it was founded no relationship between each of the markers studied and the occurrence of mammary tumor between the problem group and the control group.

Therapy-induced senescence promotes breast cancer cells plasticity by inducing Lipocalin-2 expression.

The acquisition of novel detrimental cellular properties following exposure to cytotoxic drugs leads to aggressive and metastatic tumors that often translates into an incurable disease. While the bulk of the primary tumor is eliminated upon exposure to chemotherapeutic treatment, residual cancer cells and non-transformed cells within the host can engage a stable cell cycle exit program named senescence. Senescent cells secrete a distinct set of pro-inflammatory factors, collectively termed the senescence-associated secretory phenotype (SASP). Upon exposure to the SASP, cancer cells undergo cellular plasticity resulting in increased proliferation, migration and epithelial-to-mesenchymal transition. The molecular mechanisms by which the SASP regulates these pro-tumorigenic features are poorly understood. Here, we report that breast cancer cells exposed to the SASP strongly upregulate Lipocalin-2 (LCN2). Furthermore, we demonstrate that LCN2 is critical for SASP-induced increased migration in breast cancer cells, and its inactivation potentiates the response to chemotherapeutic treatment in mouse models of breast cancer. Finally, we show that neoadjuvant chemotherapy treatment leads to LCN2 upregulation in residual human breast tumors, and correlates with worse overall survival. These findings provide the foundation for targeting LCN2 as an adjuvant therapeutic approach to prevent the emergence of aggressive tumors following chemotherapy.

Synthesis, X-ray crystal structure, DFT calculation and anti-tumor research of novel-configurational dihydroartemisinin purine hybrids

The diastereomers 10-N-[2-fluoro-6-chloro-9H-9-purinyl]-(9R, 10R)-dihydroartemisinin(A) and10-N-[2-fluoro-6-chloro-9H-9-purinyl]-(9S, 10S)-dihydroartemisinin(B) were obtained via a trifluoroacetic acid catalyzed procedure simultaneously. Their structures were characterized by HRMS, MS, 1H-NMR, 13C-NMR, 2D NMR(COSY) and IR spectroscopy, and the configurations were further confirmed by X-Ray diffraction and Density functional theory (DFT). Then, the intermolecular interactions were studied mainly through Hershfield surface (HS), molecular electrostatic potential (MEP) and frontier molecular orbital (FMO) methods. Finally, preliminary anticancer evaluations of A and B were conducted in human breast tumor cell lines (MDA-MB-436, T47D) and a normal mammary epithelial cell line (MCF-10A). The GI50 of A and B were better or comparable to the positive drug mercaptopurine (6-MP), and both of novel compounds did not display significant cytotoxicity toward MCF-10A.

Andrographolide suppresses breast cancer progression by modulating tumor-associated macrophage polarization through the Wnt/β-catenin pathway.

Andrographolide(ADE) has been demonstrated to inhibit tumor growth through direct cytotoxicity on tumor cells. However, its potential activity on tumor microenvironment (TME) remains unclear. Tumor-associated macrophages (TAMs), composed mainly of M2 macrophages, are the key cells that create an immunosuppressive TME by secretion of cytokines, thus enhancing tumor progression. Re-polarized subpopulations of macrophages may represent vital new therapeutic alternatives. Our previous studies showed that ADE possessed anti-metastasis and anoikis-sensitization effects. Here, we demonstrated that ADE significantly suppressed M2-like polarization and enhanced M1-like polarization of macrophages. Moreover, ADE inhibited the migration of M2 and tube formation in HUVECs under M2 stimulation. In vivo studies showed that ADE restrained the growth of MDA-MB-231 and HCC1806 human breast tumor xenografts and 4T-1 mammary gland tumors through TAMs. Wnt5a/β-catenin pathway and MMPs were particularly associated with ADE's regulatory mechanisms to M2 according to RNA-seq and bioinformatics analysis. Moreover， western blot also verified the expressions of these proteins were declined with ADE exposure. Among the cytokines released by M2, PDGF-AA and CCL2 were reduced. Our current findings for the first time elucidated that ADE could modulate macrophage polarization and function through Wnt5a signaling pathway, thereby playing its role in inhibition of triple-negative breast cancer.

Low lamin A levels enhance confined cell migration and metastatic capacity in breast cancer.

Aberrations in nuclear size and shape are commonly used to identify cancerous tissue. However, it remains unclear whether the disturbed nuclear structure directly contributes to the cancer pathology or is merely a consequence of other events occurring during tumorigenesis. Here, we show that highly invasive and proliferative breast cancer cells frequently exhibit Akt-driven lower expression of the nuclear envelope proteins lamin A/C, leading to increased nuclear deformability that permits enhanced cell migration through confined environments that mimic interstitial spaces encountered during metastasis. Importantly, increasing lamin A/C expression in highly invasive breast cancer cells reflected gene expression changes characteristic of human breast tumors with higher LMNA expression, and specifically affected pathways related to cell-ECM interactions, cell metabolism, and PI3K/Akt signaling. Further supporting an important role of lamins in breast cancer metastasis, analysis of lamin levels in human breast tumors revealed a significant association between lower lamin A levels, Akt signaling, and decreased disease-free survival. These findings suggest that downregulation of lamin A/C in breast cancer cells may influence both cellular physical properties and biochemical signaling to promote metastatic progression.

Hydrophilic platinum(II) complexes containing 5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidine: Spectroscopic and in vitro cytotoxic characterization

Mononuclear platinum(II) complexes with 5,7-methyl-1,2,4-triazolo[1,5-a]pyrimidine (dmtp) of the general formula cis-[Pt(X)2(dmtp)2], where X - acetate (1), trichloroacetate (2), trifluoroacetate (3), nitrate (4) have been synthesized and characterized by multinuclear magnetic resonance (1H, 13C, 15N, 195Pt) and infrared spectroscopy methods. Spectroscopic parameters indicated the presence of the PtO2N2 chromophore system with two monodentate N(3)-bonded 5,7-methyl-1,2,4-triazolo[1,5-a]pyrimidines, and two monodentate O-donor ligands (carboxylate or nitrate).A cytotoxicity assay of hydrophilic platinum(II) complexes (1–4) against three human tumour cell lines A549 (non-small cell lung carcinoma), T47D (breast cancer), HT-29 (colon adenocarcinoma), and normal murine embryonic fibroblast cells (BALB/3T3) was performed.Considering the cytotoxic parameters of the studied complexes, the best in vitro results against the human breast tumour cells (T47D) were found for cis-[Pt(OOCCCl3)2(dmtp)2] although all the tested complexes (except complex (4)) exhibited moderate in vitro activity.

Upregulation of Nox4 induces a pro-survival Nrf2 response in cancer-associated fibroblasts that promotes tumorigenesis and metastasis, in part via Birc5 induction

BackgroundA pro-oxidant enzyme, NADPH oxidase 4 (Nox4) has been reported to be a critical downstream effector of TGFβ-induced myofibroblast transformation during fibrosis. While there are a small number of studies suggesting an oncogenic role of Nox4 derived from activated fibroblasts, direct evidence linking this pro-oxidant to the tumor-supporting CAF phenotype and the mechanisms involved are lacking, particularly in breast cancer.MethodsWe targeted Nox4 in breast patient-derived CAFs via siRNA-mediated knockdown or administration of a pharmaceutical inhibitor (GKT137831). We also determine primary tumor growth and metastasis of implanted tumor cells using a stable Nox4-/- syngeneic mouse model. Autophagic flux of CAFs was assessed using a tandem fluorescent-tagged ptfl-LC3 plasmid via confocal microscopy analysis and determination of the expression level of autophagy markers (beclin-1 and LC3B). Nox4 overexpressing CAFs depend on the Nrf2 (nuclear factor-erythroid factor 2-related factor 2) pathway for survival. We then determined the dependency of Nox4-overexpressing CAFs on the Nrf2-mediated adaptive stress response pathway for survival. Furthermore, we investigated the involvement of Birc5 on CAF phenotype (viability and collagen contraction activity) as well as the expression level of CAF markers, FAP and αSMA.ConclusionsWe found that deletion of stroma Nox4 and pharmaceutically targeting its activity with GKT137831 significantly inhibited orthotopic tumor growth and metastasis of implanted E0771 and 4T1 murine mammary carcinoma cell lines in mice. More importantly, we found a significant upregulation of Nox4 expression in CAFs isolated from human breast tumors versus normal mammary fibroblasts (RMFs). Our in situ RNA hybridization analysis for Nox4 transcription on a human breast tumor microarray further support a role of this pro-oxidant in the stroma of breast carcinomas. In addition, we found that Nox4 promotes autophagy in CAFs. Moreover, we found that Nox4 promoted survival of CAFs via activation of Nrf2, a master regulator of oxidative stress response. We have further shown Birc5 is involved as a downstream modulator of Nrf2-mediated pro-survival phenotype. Together these studies indicate a role of redox signaling via the Nox4-Nrf2 pathway in tumorigenesis and metastasis of breast cancer cells by promoting autophagy and survival of CAFs.

In vitro cytotoxic and antioxidant evaluation of quercetin loaded in ionic cross-linked chitosan nanoparticles

The objective of this work is the nanoencapsulation of quercetin in chitosan (CHI) nanoparticles (NPs) cross-linked by ionic gelation with sodium tripolyphosphate (TPP), in order to study the release profile of quercetin and evaluate its in vitro cytotoxic and antioxidant activity using tumor cells and the DPPH method, respectively. The NPs synthesized by ionic gelation method showed a low polydispersity index (PDI) of 0.208 ± 0.03, hydrodynamic diameter of 103.2 ± 5.7 nm and positive zeta potential with high module value (+30.4 ± 1.6 mV). These characteristics were directly influenced by the variation of the TPP volume. The NPs showed good colloidal stability during the period of 90 days at temperature of 37 °C. Samples were also characterized by DLS, BET, FTIR, TEM and TGA. The encapsulation efficiency of the quercetin in CHI/TPP NPs was 83.8 ± 0.33%, and its release was gradual and faster in solutions of NaH2PO4 at pH 7.4 by non-Fickian mechanism. From the evaluation of antioxidant activity in vitro using the DPPH method, it was observed that CHI/TPP/Quercetin NPs showed greater antioxidant effect when compared to free quercetin. Using the 3,4,5-dimethylthiazol-2,5 biphenyl tetrazolium bromide (MTT) assay, we observed that quercetin loaded in NPs showed a significant cytotoxic effect against human breast tumor (MCF-7) and human lung tumor (A549) in the 72-h period.

Natural Compounds or Their Derivatives against Breast Cancer: A Computational Study.

Background Breast cancer is one of the most common types of cancer diagnosed and the second leading cause of death among women. Breast cancer susceptibility proteins of type 1 and 2 are human tumor suppressor genes. Genetic variations/mutations in these two genes lead to overexpression of human breast tumor suppressor genes (e.g., BRCA1, BRCA2), which triggers uncontrolled duplication of cells in humans. In addition, multidrug resistance protein 1 (MDR1), an important cell membrane protein that pumps many foreign substances from cells, is also responsible for developing resistance to cancer chemotherapy. Aim of the Study. The aim of this study was to analyze some natural compounds or their derivatives as part of the development of strong inhibitors for breast cancer. Methodology. Molecular docking studies were performed using compounds known in the literature to be effective against BRCA1 and BRCA2 and MDR1, with positive control being 5-fluorouracil, an antineoplastic drug as a positive control. Results The binding affinity of the compounds was analyzed, and it was observed that they had a better binding affinity for the target proteins than the standard drug 5-fluorouracil. Among the compounds analyzed, α-hederin, andrographolide, apigenin, asiatic acid, auricular acid, sinularin, curcumin, citrinin, hispolon, nerol, phytol, retinol palmitate, and sclareol showed the best binding affinity energy to the BRCA1, BRCA2, and MDR1 proteins, respectively. Conclusions α-Hederin, andrographolide, apigenin, asiatic acid, auricular acid, hispolon, sclareol, curcumin, citrinin, and sinularin or their derivatives can be a good source of anticancer agents in breast cancer.

Rescue of a peroxisome proliferator activated receptor gamma gene network in muscle after growth of human breast tumour xenografts

AbstractBackgroundFatigue is common in patents with breast cancer (BC), and can occur in patients with early stage disease and in the absence of muscle wasting (i.e. cachexia). We have reported transcriptional and proteomic alterations in muscles from BC patients, which are associated with fatigue. Mice implanted with human BC xenografts recapitulate the muscle molecular composition changes seen in patients, coupled with a greater rate of contraction‐induced fatigue. Multiple bioinformatics platforms in both human and mouse muscles have identified peroxisome proliferator activated receptor gamma (PPARG) as central to this phenotype, with several PPARG target genes downregulated in muscle in response to tumour growth. The current study tested the hypothesis that the PPARG agonist pioglitazone (pio), a commonly prescribed diabetes drug, would rescue the transcriptional alterations observed in muscles of tumour‐bearing mice.MethodsSixteen female NSG mice were implanted with breast cancer patient‐derived orthotopic xenografts (BC‐PDOX) via transplantation of Her2/neu+ human tumour fragments. BC‐PDOX mice were randomly assigned to a treatment group that received daily oral pio at 30 mg/kg (n = 8), or a control group that received a similar volume of vehicle (n = 8). Treatment was initiated when tumours reached a volume of 600 mm3, and lasted for 2 weeks. Hindlimb muscles were isolated from BC‐PDOX and non‐tumour bearing mice for RNA‐sequencing, gene expression validation, and ATP quantification. Differentially expressed genes (DEGs) in muscles from BC‐PDOX mice relative to non‐tumour bearing controls were identified using DESeq2, and multiple bioinformatics platforms were employed to contextualize the DEGs.ResultsWe found that the administration of pio restored the muscle gene expression patterns of BC‐PDOX mice to a profile resembling muscles of non‐tumour bearing NSG control mice. Validation of skeletal muscle gene expression by qPCR confirmed pio increased the expression of PPARG target genes (P < 0.05 in all genes but PPARG P = 0.684) in skeletal muscles. Isolated mitochondria from muscles of BC‐PDOX mice treated with pio contained greater levels of ATP (mean luminescence 33770 ± 4057 in BC‐PDOX vs. 62780 ± 11510 in BC‐PDOX PIO, P = 0.045). There were no differences in body weights (P = 0.560), muscle weights (Gas P = 0.295, Sol P = 0.365, EDL P = 0.182, TA P = 0.415) or tumour volumes (P = 0.885) in pio versus vehicle treated BC‐PDOX mice.ConclusionsThese data demonstrate that oral pio supplementation rescues the BC‐associated downregulation of PPARG target genes in skeletal muscle. Additionally, muscles from BC‐PDOX mice treated with pio had greater levels of ATP, which would be associated a more fatigue‐resistant muscle phenotype. Therefore, we propose that the FDA‐approved and generic diabetes drug, pio, be considered as a supportive therapy for the treatment of BC‐associated muscle fatigue.

Functional Restoration of BRCA1 Nonsense Mutations by Aminoglycoside-Induced Readthrough.

BRCA1 is a major tumor suppressor that functions in the accurate repair of DNA double-strand breaks via homologous recombination (HR). Nonsense mutations in BRCA1 lead to inactive truncated protein products and are associated with high risk of breast and ovarian cancer. These mutations generate premature termination codons (PTCs). Different studies have shown that aminoglycosides can induce PTC suppression by promoting stop codon readthrough and restoring full-length (FL) protein expression. The use of these compounds has been studied in clinical trials for genetic diseases such as cystic fibrosis and Duchenne muscular dystrophy, with encouraging results. Here we show proof-of-concept data demonstrating that the aminoglycoside G418 can induce BRCA1 PTC readthrough and restore FL protein synthesis and function. We first demonstrate that G418 treatment restores BRCA1 FL protein synthesis in HCC1395, a human breast tumor cell line carrying the R1751X mutation. HCC1395 cells treated with G418 also recover HR DNA repair and restore cell cycle checkpoint activation. A set of naturally occurring BRCA1 nonsense variants encoding different PTCs was evaluated in a GFP C-terminal BRCA1 construct model and BRCA1 PTC readthrough levels vary depending on the stop codon context. Because PTC readthrough could generate FL protein carrying pathogenic missense mutations, variants representing the most probable acquired amino acid substitutions in consequence of readthrough were functionally assessed by a validated transcription activation assay. Overall, this is the first study that evaluates the readthrough of PTC variants with clinical relevance in the breast and ovarian cancer-predisposing gene BRCA1.

Activation Versus Inhibition of IGF1R: A Dual Role in Breast Tumorigenesis.

Historically, the body of literature surrounding the insulin-like growth factor type 1 receptor (IGF1R) has described a largely pro-tumorigenic role in breast cancer cells and in several transgenic or xenograft mouse models of breast cancer. Interestingly, however, more recent evidence has emerged that suggests an additional, previously undescribed, tumor and metastasis suppressive function for IGF1R in both human breast tumors and mammary oncogenesis in mice. These seemingly conflicting reports can be reconciled when considering what is currently known about IGF1R function in the context of tissue development and cancer as it relates to cellular growth, proliferation, and differentiation. In this mini review, we will summarize the currently existing data with a particular focus on mouse models that have been developed to study IGF1R function in mammary development, tumorigenesis, and metastasis in vivo and propose hypotheses for how both the tumor-promoting and tumor-suppressing schools of thought regarding IGF1R in these histological contexts are compatible.

Abstract 834: Serine protease hepsin regulates tumor growth via TGFbeta-EGFR signaling axis

Abstract Hepsin (encoded by HPN gene) is a type II transmembrane serine protease, which is commonly overexpressed in carcinomas of prostate and breast. Hepsin protein is known to be stabilized by Ras-MAPK pathway signaling, and downstream, this protease regulates the degradation of extracellular matrix (ECM) components and activates growth factor pathways, including hepatocyte growth factor (HGF) and transforming growth factor beta (TGF beta) pathway. However, the impact of the hepsin-dependent signaling on cell proliferation and tumor growth is not well-understood. Therefore, we sought to clarify the role of hepsin during these events using engineered breast cancer cell lines, Hpn CRISPR knockout mouse model crossed with Wap-Myc breast cancer mouse model, and patient-derived explant cultures (PDEC)s from human breast tumors. We isolated Wap-Myc; Hpn-/- mammary tumor cells and made orthotopic transplantation into syngeneic wild-type recipient mice. The resulting Wap-Myc tumors that lack hepsin had reduced size both in primary and metastatic site compared to tumors derived from Wap-Myc; Hpn+/+ tumor cells. This decrease in growth was accompanied by downregulation in TGF beta and EGFR signaling as well as substantial reduction in total EGFR protein level. We further demonstrated that in 3D culture conditions overexpression of hepsin induced cell proliferation, which was TGF beta 1 and EGFR signaling-dependent, while PDECs treated with hepsin inhibitory antibodies and small molecules had decreased EGFR and TGF beta signaling activity and reduction in proliferation marker expression. Taken together, this study demonstrates a role for hepsin as a regulator of cell proliferation and tumor growth through TGF beta and EGFR pathways and may provide an interesting druggable upstream target to inhibit TGF beta and EGFR pathways in breast cancer. Citation Format: Topi A. Tervonen, Denis Belitškin, Pauliina Munne, Shishir M. Pant, Ilida Suleymanova, Kati Belitškina, Jeroen Pouwels, Juha Klefström. Serine protease hepsin regulates tumor growth via TGFbeta-EGFR signaling axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 834.

Abstract 2423: A Cdh3 - Lam332 signaling axis by a unique subset of leader cells controls breast tumor organoid protrusion dynamics and directed collective migration

Abstract Carcinoma dissemination can occur when heterogeneous tumor and tumor stromal cells clusters migrate together via collective migration. Cells at the front lead and direct collective migration, yet how these leader cells form and interact with the microenvironment to direct migration are not fully appreciated. From live videos of primary mouse and human breast tumor organoids in a 3D microfluidic system that mimics the native breast tumor microenvironment, we developed 3D computational models. These hypothesize that for leader cells to polarize to the leading edge and lead directed collective migration they need to generate high protrusive forces and overcome extracellular matrix (ECM) resistance. From scRNAseq of migrating primary tumor organoids, we identify significant K14+ “leader” cell heterogeneity that differs depending upon the environmental signal. We isolate a unique Cadherin-3 (Cdh3) positive leader cell subpopulation that is necessary and sufficient to lead collective migration. Cdh3 controls leader cell protrusion dynamics through controlling the local production of Laminin-332 which is required for integrin/focal adhesion function. Loss of Cdh3 expression in mouse and human primary breast tumor organoids and invasive breast tumor cell lines significantly impairs directed collective migration. In the absence of leader cell Cdh-Cdh3 intercellular adhesions there is reduced cellular β-catenin levels and Laminin 332 production. In syngeneic, orthotopic transplant mouse models, Cdh3 depletion in tumor cells resulted in decreased laminin 332 production at the invasive edge of primary tumors and decreased lung metastasis. Chromatin Immunoprecipitation experiments revealed that in contacted cells β-catenin and TCF4 are present on Lam α3, β3, and γ2 promoter regions. Laminin 332 production by leader cells was required for optimal Integrin/FA function and cellular protrusion stability. These results indicated that in leader cells local Cdh3-Cdh3/β-catenin regulated Laminin 332 production controls protrusion dynamics and overcomes resistance to ECM to lead directed collective tumor migration. Our findings highlight how a unique subset of leader cells in breast tumors interact with the microenvironment to direct collective migration. Citation Format: Yanyang Cao, Priscilla Y. Hwang, Maria Clarke, Jose Almeida, Amit Pathak, Gregory D. Longmore. A Cdh3 - Lam332 signaling axis by a unique subset of leader cells controls breast tumor organoid protrusion dynamics and directed collective migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2423.

Abstract 3865: Spatial multiplex profiling of immune cell markers in FFPE tumor tissues using the RNAscope™ HiPlex v2 in situ hybridization assay

Abstract The tumor microenvironment (TME) is highly complex, comprised of tumor cells, immune cells, stromal cells, and extracellular matrix. Understanding spatial interactions between various cell types and their activation states in the TME is crucial for implementing successful immunotherapy strategies against various types of cancer. This study demonstrates a highly sensitive and specific multiplexed technique, the RNAscope HiPlex v2 in situ hybridization (ISH) assay for spatial and transcriptomic profiling of target genes to assess immune regulation in human lung, breast, cervical and ovarian FFPE tumor tissues. We have expanded our current RNAscope HiPlex assay capability of iteratively multiplexing up to 12 targets in fixed and fresh frozen samples to include formalin fixed paraffin embedded (FFPE) tissues. The novel FFPE reagent effectively reduces background autofluorescence, improving the signal to noise ratio. We have leveraged this technology to investigate spatial expression of 12 oncology and immuno-oncology target genes, including tumor markers, immune checkpoint markers, immunosuppression markers, immune cell markers and secreted chemokine RNA expression profile within the TME. The targets were simultaneously registered using HiPlex image registration software v2 that enables background subtraction. We visualized T cell infiltration and identified T cell subsets within tumors using CD3 and CD8 expression and activated T cells by IFNG expression. We further identified subsets of pro- and anti-inflammatory macrophages by CD68 and CD163 expression as well effector cells which secrete chemokines and cytokine. We also detected the hypoxia markers HIF1A and VEGF to elucidate the immunosuppressive state of tumor cells. Preliminary analysis and quantification of the HIF1A expression using HALO® image analysis software showed higher copy numbers in the lung tumor as compared to the other tumors, demonstrating the sensitivity of the assay through differential expression. We additionally showed the differential expression of immune checkpoint markers PDCD1, and CD274 within the TME. Using a highly sensitive multiplexed RNAscope HiPlex v2 ISH assay, we have demonstrated the capability of this technique to spatially resolve 12 targets in four different tumor types. The FFPE reagent efficiently quenched background autofluorescence in the tissues and identified immune cell signatures within the TME. Quantification of immunosuppressive markers further depicted differential expression among various tumors. This technology is highly beneficial for investigating complex and spatial tumor-stroma interactions in basic science and translational research. The assay can also provide valuable understanding of the biological crosstalk among various cell types in complex and heterogeneous tissues. Citation Format: Anushka Dikshit, Sayantani Basak, Ching-Wei Chang, Michaeline Bunting, Kim Collins. Spatial multiplex profiling of immune cell markers in FFPE tumor tissues using the RNAscope™ HiPlex v2 in situ hybridization assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3865.

Abstract 5030: Intra-patient tumor evolution analysis in dogs with many mammary tumors identifies signatures of tumor aggression in early-stage breast cancers

Abstract Despite tremendous improvements in breast cancer detection, it remains unclear which early-stage tumors will later become aggressive. Dogs provide a unique opportunity to unravel early-stage tumor evolution, as dogs frequently develop multiple naturally occurring mammary tumors. To identify which early-stage human breast tumors will later become malignant, we analyzed mutational profiles of mammary tumors from two canine cohorts to identify patterns of tumor evolution. We compared our dog tumor signatures with human breast cancers. We found that dogs and humans share many known cancer driver mutations. Additionally, three of the five dogs with at least five mammary tumors had a single driver mutation present in most of their tumors. Despite sharing patient-level exposures and environment, passenger mutations significantly differ between these tumors, suggesting independent primaries initiated by a catalyzing factor causing the same driver mutation in each tumor. However, tumors from the same dog with the same driver gene mutations rarely exhibit the same expression-based breast cancer subtypes. Our analysis highlights the mutational similarities of dog and human breast cancers and provides insight into their development. Citation Format: Kiley Graim. Intra-patient tumor evolution analysis in dogs with many mammary tumors identifies signatures of tumor aggression in early-stage breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5030.

Abstract 3127: Negative impact of paclitaxel on human breast tumor microenvironment and its reversal by the combination of interferon-α with TLR3 agonist rintatolimod

Abstract Background: Taxanes are extensively used in the chemotherapy of breast cancer in the neoadjuvant, adjuvant and metastatic settings. Paclitaxel is known to have both immunostimulatory and immunosuppressive effects, but its impact on the chemokine system within the tumor microenvironment (TME) remains unclear. In the breast TME treated with neoadjuvant taxane chemotherapy, high levels of the cytotoxic T cell (CTL) attractants CXCL9, CXCL10 and their common receptor CXCR3 predict higher probability of pathological complete response (pCR) and improved long term outcomes. We previously developed a chemokine modulatory regimen (CKM) combining interferon-α with TLR3 agonist rintatolimod to selectively increase CTL-attracting chemokines in the TME but reduce the production of chemokines attracting myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) in the TME. Here we evaluated paclitaxel’s impact on chemokine production in the human breast TME and CKM’s ability to eliminate undesirable aspects of taxane chemotherapy. Methods: Fresh breast cancer tissues obtained during routine surgeries, breast cancer cell lines BT-549 and MDA-MD-231 and peripheral blood monocyte-derived macrophages were analyzed immediately or cultured ex vivo for 24 hours in the absence or presence of paclitaxel and/or CKM components. The expression of chemokine genes and secretion of chemokines by freshly harvested and ex vivo-treated tumor explants, cancer cell lines and macrophages were analyzed by quantitative PCR (Taqman) and ELISA. Paired student t-test was used for statistical analyses. Results: Breast cancer explants spontaneously expressed high levels of MDSC/Treg-attractants CXCL12 and CCL22, but only marginal levels of CTL and natural killer (NK) cell attractants CXCL9, CXCL10, CXCL11 and CCL5. Unexpectedly, paclitaxel treatment resulted in further elevation of granulocyte/MDSC-attractant CXCL8 and CCL22, which was reversed by the combination of paclitaxel with CKM. At the same time, while paclitaxel alone did not induce any of the CTL attractants tested, its combination with the CKM was highly effective in inducing CXCL9, CXCL10, CXCL11 and CCL5. Conclusions: Our results identify an undesirable aspect of paclitaxel’s impact on breast cancer. The ability of CKM to enhance the expression of CTL/NK cell attractants and suppress the production of Treg/MDSC attractants produced by paclitaxel provides a strong rationale for combined use of CKM in taxane-based chemo-immunotherapy of breast cancer and potentially other diseases. Citation Format: Shipra Gandhi, Melissa Grimm, Ronald Slomba, Kazuaki Takabe, Pawel Kalinski. Negative impact of paclitaxel on human breast tumor microenvironment and its reversal by the combination of interferon-α with TLR3 agonist rintatolimod [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3127.

Abstract 5813: Glycogen synthesis as potential novel target in triple negative breast cancer: Glycogen synthase 1 expression in human breast cancers and the impact of downregulation on proliferation of preclinical models

Abstract Background: Triple-negative breast cancer (TNBC) has a poor clinical prognosis and is characterized by a lack of druggable targets and a hypoxic tumor microenvironment. Hypoxia-induced glycogen accumulation and utilization are involved in cancer proliferation and therapy resistance, making modulation of glycogen metabolism of therapeutic interest. Therefore, we studied expression of glycogen synthase 1 (GYS1, the key regulator of glycogen synthesis) and glycogen stores in publicly available expression data and human breast tumors including TNBC. Also, we studied downregulation of GYS1 in preclinical breast cancer models, focusing on TNBC. Methods: GYS1 mRNA expression and correlations with survival per clinical subtype were assessed in the METABRIC dataset. A tissue micro-array was constructed from primary tumors of 396 breast cancer patients with long-term follow-up, including normal breast control tissue. Triplicate tissue cores were stained immunohistochemically for GYS1, glycogen and the hypoxic marker carbonic anhydrase 9, and with periodic acid-Schiff staining for glycogen. In four TNBC cell lines and an MDA-MB-231 xenograft model, GYS1 protein expression, glycogen content and cell proliferation in normoxia and hypoxia were evaluated +/- GYS1 knockdown by siRNA or shRNA. Sensitivity of shGYS1 cell lines to drugs targeting mitochondria was tested. Results: In the METABRIC dataset (n = 1904), overexpression of GYS1 mRNA was associated with poor overall survival (HR 1.20 [95% CI 1.05 - 1.38]), which was mainly driven by the TNBC patients (n = 299, HR 1.52 [95% CI 1.09 - 2.14]). Immunohistochemically, most primary breast tumors had elevated GYS1 levels and glycogen content compared to normal breast tissue, with subtype specific analyses ongoing. In all breast cancer cell lines, hypoxia induced GYS1 protein expression and increased glycogen content. Acute siRNA-mediated GYS1 knockdown decreased proliferation of (TN)BC cell lines and MDA-MB-231 spheroids in both hypoxia and normoxia. shGYS1 MDA-MB-231 cells had decreased glycogen levels and shGYS1 MDA-MB-231 xenograft growth was impaired, especially in the first six weeks after inoculation. In control xenografts, immunohistochemical GYS1 expression was most pronounced adjacent to the necrotic tumor core, whereas shGYS1 xenografts lacked GYS1 expression. Finally, shGYS1 MDA-MB-231 cells were more sensitive to inhibitors of mitochondrial protein homeostasis, suggesting a potential synergistic approach to overcome eventual metabolic adaptation. Conclusions: GYS1 is overexpressed in primary breast tumors and high mRNA levels correlate with poor survival in TNBC. GYS1 downregulation impairs TNBC proliferation in vitro and in vivo, highlighting glycogen synthesis as potential novel therapeutic target in TNBC. Citation Format: Ellen C. De Heer, Christos E. Zois, Esther Bridges, Mieke C. Zwager, Tineke van der Sluis, Bert van der Vegt, Carolien P. Schröder, Steven de Jong, Adrian L. Harris, Mathilde Jalving. Glycogen synthesis as potential novel target in triple negative breast cancer: Glycogen synthase 1 expression in human breast cancers and the impact of downregulation on proliferation of preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5813.

Mitophagy Induction and Aryl Hydrocarbon Receptor-Mediated Redox Signaling Contribute to the Suppression of Breast Cancer Cell Growth by Taloxifene via Regulation of Inflammasomes Activation.

Aims: Raloxifene, a selective estrogen receptor (ER) modulator, has been reported to exert the tumor-suppressive effects in both ER-positive and ER-negative cancer cells; however, the mechanisms underlying its ER-independent anti-cancer effects are poorly understood. The NLRP3 inflammasome, a critical component of the innate immune system, has recently received growing attention owing to its multifaceted roles in various aspects of cancer development. The present study aimed at examining the involvement of NLRP3 inflammasomes in the anti-breast cancer effects of raloxifene and its underlying mechanisms. Results: Raloxifene significantly inhibited the activation of NLRP3 inflammasomes in various breast cancer cell lines. Importantly, forced expression of a gain-of-function variant of NLRP3 rescued breast cancer cells from growth arrest by raloxifene, suggesting that the suppression of NLRP3 inflammasomes activation mediates the raloxifene-induced inhibition of breast cancer growth. Mechanistically, raloxifene suppressed NLRP3 inflammasomes activation by lowering the cellular levels of reactive oxygen species (ROS) through the modulation of redox signaling mediated via aryl hydrocarbon receptor (AhR)-nuclear factor erythroid 2-related factor 2 (Nrf2)-heme oxygenase-1 (HO-1) axis or the impaired generation of mitochondrial ROS in a mitophagy-dependent manner. Further, the blockage of AhR signaling or inhibition of mitophagy abolished the tumor-suppressive effect of raloxifene in a human breast tumor xenograft model. Innovation: We elucidate a novel molecular mechanism underlying the breast tumor suppressing effect of raloxifene. Conclusion: The results observed in this study suggest that the modulation of NLRP3 inflammasomes activation is a critical event in the inhibition of breast tumor growth by raloxifene. Antioxid. Redox Signal. 37, 1030-1050.