Human Ductal Carcinoma Research Articles

Low progesterone receptor levels in high-grade DCIS correlate with HER2 upregulation and the presence of invasive components.

In this study, we investigated pivotal molecular markers in human high-grade breast ductal carcinoma in situ (DCIS). Expression status of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth receptor 2 (HER2) was measured among various subtypes (Luminal (Lum) A, LumB HER2-, LumB HER2+, HER2-enriched and triple-negative). In total, 357 DCIS cases were classified into respective subtypes, according to the 2013 St. Gallen guidelines. Each subtype was categorized into three subcategories: "Pure" (those without an invasive component), "W/invasive" (those with an invasive component), and "All" (the entire group of the given subtype). ER and PR expression were registered as intervals. Equivocal HER2 immunohistochemistry (IHC) cases (2+) were further investigated using dual-color in situ hybridization. The majority of patients (71%) were over the age of 50. We discovered no significant differences in the proportion of age between the "Pure" and "W/invasive" groups. There was no significant difference in ER/PR expression between "Pure" luminal subtypes of DCIS and "W/invasive" cases. We compared the HER2 IHC scores of "0", "1+", and "2+" among LumA and LumB HER2 subtypes and identified no statistically significant differences between "Pure" and "W/invasive" (p = 0.603). ER and PR expression ≥ 50% cutoff value was present in > 90% of all LumA cases. The incidences of cases with ER expression at cutoff values of < 10% and ≥ 50% in LumA were significantly different compared to other luminal subtypes (p < 0.0001). The proportion of cases with PR expression < 20% showed significant differences in the various luminal subtypes. In luminal B subtypes, low PR expression (< 20%) was significantly associated with both strong HER2 expression (3+) and the presence of an invasive component (p = 0.0001 and p = 0.0365, respectively). ER and PR expression at ≥ 50% cutoff values were found in more than 90% of LumA cases. Samples with ER < 10% and ≥ 50% in LumA were significantly different compared to other luminal subtypes (p < 0.0001). Low PR expression in high-grade DCIS was strongly associated with HER2 overexpression (3+) and an invasive component (p = 0.0001 and p = 0.0365, respectively).

The functional role of Nudt2 in human triple negative breast cancer.

The main known function of Nudix hydrolase 2 (Nudt2) is to hydrolyze the secondary messenger diadenosine 5', 5'''-p1, p4-tetraphosphate (Ap4A). In this study we examined the role of Nudt2 in breast carcinoma through its expression in human invasive ductal carcinoma tissues, and its functions in human triple negative breast cancer (TNBC) cell lines. A significantly higher expression of Nudt2 was observed in human invasive ductal carcinoma tissues compared to that in normal breast tissue. Knockdown of Nudt2 in TNBC cell lines resulted in a significant reduction in cellular proliferation via the Ki67 marker, accompanied by G0/G1 phase cell cycle arrest, in the migration and invasion of these cells and in tumorigenicity and anchorage-independent growth. It can therefore be concluded that Nudt2 plays a significant role in promoting TNBC growth.

Abstract LB365: Generation of a novel severely immunodeficient Sprague Dawley rat model for xenograft studies

Abstract The recombinant-activating gene 2 (Rag2) plays a critical role in the recombination of the immunoglobulin and T cell receptor genes during the maturation of T and B cells. Loss of Rag2 protein leads to a lack of functional T and B cells, which eliminates the adaptive immune response. However, Rag2 deficiency alone may not guarantee the success of xenograft implantation, due to NK cells and other innate cells remaining. Il2rg is a key component of several cytokine receptors involved in differentiation and/or function of numerous adaptive and innate immune cells, including NK cells, mast cells, basophils, and dendritic cells. To generate a more severely immunodeficient rat model, both the Rag2 and Il2rg genes were knocked out in Sprague Dawley (SD) rats; this model was termed B-SDG rats. The immune profile and organ weights of thymus and spleen were investigated in B-SDG rats in comparison with Rag2-/- and wild-type SD rats. The spleen weights of both Rag2-/- and B-SDG rats were similar, but significantly reduced compared to wild-type SD rats. Notably, the thymus weights of B-SDG male rats were significantly smaller than those of Rag2-/- males. The immune cell profiles in thymus, spleen and blood were next evaluated by flow cytometry. A complete lack of B and T cells was observed in both Rag2-/- and B-SDG rats. B-SDG rats lacked CD161+ NK cells in the spleen and blood, which were maintained in Rag2-/- rats. Dendritic cells, monocytes, macrophages, and neutrophils were detected in all strains. In vivo xenograft implantation with human lung, bladder, breast, pancreas, acute monocytic and myelomonocytic leukemia tumor cell lines was next evaluated in B-SDG rats, which demonstrated successful engraftment. Furthermore, several efficacy studies were performed in B-SDG rats, including cisplatin chemotherapy on human breast invasive ductal carcinoma cell (HCC1954) and human large cell lung cancer cell (NCI-H460) xenografts, and a HER2-targeting ADC therapy was tested in B-SDG rats harboring human non-small cell lung cancer tumors (NCI-H1975). Taken together, these data confirm that B-SDG rats provide a powerful immunodeficient model for preclinical in vivo evaluation of novel drug candidates. Citation Format: Yanhui Nie, Meiqi Zhang, Wanling Zhong, Jia Feng, Bahetiyaer Huwatibieke, Zhaoxue Yu. Generation of a novel severely immunodeficient Sprague Dawley rat model for xenograft studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB365.

SpatialcoGCN: deconvolution and spatial information-aware simulation of spatial transcriptomics data via deep graph co-embedding.

Spatial transcriptomics (ST) data have emerged as a pivotal approach to comprehending the function and interplay of cells within intricate tissues. Nonetheless, analyses of ST data are restricted by the low spatial resolution and limited number of ribonucleic acid transcripts that can be detected with several popular ST techniques. In this study, we propose that both of the above issues can be significantly improved by introducing a deep graph co-embedding framework. First, we establish a self-supervised, co-graph convolution network-based deep learning model termed SpatialcoGCN, which leverages single-cell data to deconvolve the cell mixtures in spatial data. Evaluations of SpatialcoGCN on a series of simulated ST data and real ST datasets from human ductal carcinoma in situ, developing human heart and mouse brain suggest that SpatialcoGCN could outperform other state-of-the-art cell type deconvolution methods in estimating per-spot cell composition. Moreover, with competitive accuracy, SpatialcoGCN could also recover the spatial distribution of transcripts that are not detected by raw ST data. With a similar co-embedding framework, we further established a spatial information-aware ST data simulation method, SpatialcoGCN-Sim. SpatialcoGCN-Sim could generate simulated ST data with high similarity to real datasets. Together, our approaches provide efficient tools for studying the spatial organization of heterogeneous cells within complex tissues.

Investigation of oncolytic potential of vaccine strains of yellow fever and tick-borne encephalitis viruses against glioblastoma and pancreatic carcinoma cell lines

Flaviviruses, possessing natural neurotropicity could be used in glioblastoma therapy using attenuated strains or as a delivery system for antitumor agents in an inactivated form. To investigate the sensitivity of glioblastoma and pancreatic carcinoma cell lines to vaccine strains of yellow fever and tick-borne encephalitis viruses. Cell lines: glioblastoma GL-6, T98G, LN-229, pancreatic carcinoma MIA RaCa-2 and human pancreatic ductal carcinoma PANC-1. Viral strains: 17D yellow fever virus (YF), Sofjin tick-borne encephalitis virus (TBEV). Virus concentration were determined by plaque assay and quantitative PCR. Determination of cell sensitivity to viruses by MTT assay. 17D YF was effective only against pancreatic carcinoma tumor cells MIA Paca-2 and had a limited effect against PANC-1. In glioblastoma cell lines (LN229, GL6, T98G), virus had no oncolytic effect and the viral RNA concentration fell in the culture medium. Sofjin TBEV showed CPE50 against MIA Paca-2 and a very limited cytotoxic effect against PANC-1. However, it had no oncolytic effect against glioblastoma cell lines (LN229, T98G and GL6), although virus reproduction continued in these cultures. For the GL6 glioblastoma cell line, the viral RNA concentration at the level with the infection dose was determined within 13 days, despite medium replacement, while in the case of the LN229 cell line, the virus concentration increased from 1 × 109 to 1 × 1010 copies/ml. Tumor behavior in organism is more complex and is determined by different microenvironmental factors and immune status. In the future, it is advisable to continue studying the antitumor oncolytic and immunomodulatory effects of viral strains 17D YF and Sofjin TBEV using in vivo models.

MTORC1 Mediates Biphasic Mechano-Response to Orchestrate Adhesion-Dependent Cell Growth and Anoikis Resistance.

Cells constantly sense and respond to not only biochemical but also biomechanical changes in their microenvironment, demanding for dynamic metabolic adaptation. ECM stiffening is a hallmark of cancer aggressiveness, while survival under substrate detachment also associates with poor prognosis. Mechanisms underlying this, non-linear mechano-response of tumor cells may reveal potential double-hit targets for cancers. Here, an integrin-GSK3β-FTO-mTOR axis is reported, that can integrate stiffness sensing to ensure both the growth advantage endowed by rigid substrate and cell death resistance under matrix detachment. It is demonstrated that substrate stiffening can activate mTORC1 and elevate mTOR level through integrins and GSK3β-FTO mediated mRNA m6 A modification, promoting anabolic metabolism. Inhibition of this axis upon ECM detachment enhances autophagy, which in turn conveys resilience of tumor cells to anoikis, as it is demonstrated in human breast ductal carcinoma in situ (DCIS) and mice malignant ascites. Collectively, these results highlight the biphasic mechano-regulation of cellular metabolism, with implications in tumor growth under stiffened conditions such as fibrosis, as well as in anoikis-resistance during cancer metastasis.

Oncolytic Activity of Canine Distemper Virus in Human Ductal Breast Carcinoma Cells

Introduction: Oncolytic virotherapy is a novel strategy for cancer treatment in humans and companion animals. Canine distemper virus (CDV) is known to induce apoptosis in tumor cells, thus serving as a potential candidate for oncolytic therapy. However, the mechanism of viral oncolytic activity is less studied and varies depending on the type of cancer and cell lines. Methods: In the present study, the susceptibility of the MCF-7 cell line to CDV infection was assessed using the CDV strain, which was confirmed previously through sequence analysis in the Vero cell line. The impact of CDV infection on cell proliferation and apoptosis was studied by evaluating the expression of four target genes including the myeloid cell leukemia 1 (MCL-1), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), transcription factor (SP1), and DNA (cytosine-5)-methyltransferase 3A (DNMT3A). Results: CDV replication in the cells induced cytopathic effect and decreased in the cell proliferation rates compared to the uninfected control. MCL-1, SP1, and PIK3R1 gene expression was down-regulated, while the expression of DNMT3A was up-regulated 3 days post-infection. The expression levels of the target genes suggest that CDV may be inducing the intrinsic apoptotic pathway in the cancer cell line. Conclusion: Overall, the results strongly propose CDV strain as a potential candidate for cancer therapy after detailed studies.

GTAD: a graph-based approach for cell spatial composition inference from integrated scRNA-seq and ST-seq data.

With the emergence of spatial transcriptome sequencing (ST-seq), research now heavily relies on the joint analysis of ST-seq and single-cell RNA sequencing (scRNA-seq) data to precisely identify cell spatial composition in tissues. However, common methods for combining these datasets often merge data from multiple cells to generate pseudo-ST data, overlooking topological relationships and failing to represent spatial arrangements accurately. We introduce GTAD, a method utilizing the Graph Attention Network for deconvolution of integrated scRNA-seq and ST-seq data. GTAD effectively captures cell spatial relationships and topological structures within tissues using a graph-based approach, enhancing cell-type identification and our understanding of complex tissue cellular landscapes. By integrating scRNA-seq and ST data into a unified graph structure, GTAD outperforms traditional 'pseudo-ST' methods, providing robust and information-rich results. GTAD performs exceptionally well with synthesized spatial data and accurately identifies cell spatial composition in tissues like the mouse cerebral cortex, cerebellum, developing human heart and pancreatic ductal carcinoma. GTAD holds the potential to enhance our understanding of tissue microenvironments and cellular diversity in complex bio-logical systems. The source code is available at https://github.com/zzhjs/GTAD.

Necroptosis-Related Prognostic Model for Pancreatic Carcinoma Reveals Its Invasion and Metastasis Potential through Hybrid EMT and Immune Escape.

Necroptosis, pro-inflammatory programmed necrosis, has been reported to exert momentous roles in pancreatic cancer (PC). Herein, the objective of this study is to construct a necroptosis-related prognostic model for detecting pancreatic cancer. In this study, the intersection between necroptosis-related genes and differentially expressed genes (DEGs) of pancreatic ductal adenocarcinoma (PDAC) was obtained based on GeneCards database, GEO database (GSE28735 and GSE15471), and verified using The Cancer Genome Atlas (TCGA). Next, a prognostic model with Cox and LASSO regression analysis, and divided the patients into high-risk and low-risk groups. Subsequently, the Kaplan-Meier (KM) survival curve and the receiver operating characteristic (ROC) curves were generated to assess the predictive ability of overall survival (OS) of PC patients. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to predict the potential biofunction and possible mechanical pathways. The EMTome database and an immune analysis were applied to further explore underlying mechanism. Finally, clinical samples of PDAC patients were utilized to verify the expression of model genes via immunohistochemistry (IHC), and the normal human pancreatic ductal cell line, hTERT-HPNE as well as human pancreatic ductal carcinoma cell lines, PANC-1 and PL45, were used to identify the levels of model genes by Western blot (WB) and immunofluorescence (IF) in vitro. The results showed that 13 necroptosis-related DEGs (NRDEGs) were screened based on GEO database, and finally four of five prognostic genes, including KRT7, KRT19, IGF2BP3, CXCL5, were further identified by TCGA to successfully construct a prognostic model. Univariate and multivariate Cox analysis ultimately confirmed that this prognostic model has independent prognostic significance, KM curve suggested that the OS of low-risk group was longer than high-risk group, and the area under receiver (AUC) of ROC for 1, 3, 5 years was 0.733, 0.749 and 0.667, respectively. A GO analysis illustrated that model genes may participate in cell-cell junction, cadherin binding, cell adhesion molecule binding, and neutrophil migration and chemotaxis, while KEGG showed involvement in PI3K-Akt signaling pathway, ECMreceptor interaction, IL-17 signaling pathway, TNF signaling pathway, etc. Moreover, our results showed KRT7 and KRT19 were closely related to EMT markers, and EMTome database manifested that KRT7 and KRT19 are highly expressed in both primary and metastatic pancreatic cancer, declaring that model genes promoted invasion and metastasis potential through EMT. In addition, four model genes were positively correlated with Th2, which has been reported to take part in promoting immune escape, while model genes except CXCL5 were negatively correlated with TFH cells, indicating that model genes may participate in immunity. Additionally, IHC results showed that model genes were higher expressed in PC tissues than that in adjacent tumor tissues, and WB and IF also suggested that model genes were more highly expressed in PANC-1 and PL45 than in hTERT-HPNE. Tracing of a necroptosis-related prognostic model for pancreatic carcinoma reveals its invasion and metastasis potential through EMT and immunity. The construction of this model and the possible mechanism of necroptosis in PDAC was preliminarily explored to provide reliable new biomarkers for the early diagnosis, treatment, and prognosis for pancreatic cancer patients.

Abstract 6350: Monoclonal antibody against BST2 isoform: potential tool for diagnostic and cancer therapy

Abstract BST2, a raft-associated type II transmembrane protein, functions as a restriction factor and inhibits HIV release by cross-linking virions onto infected cell surfaces. It also engages ILT7, a specific inhibitory receptor on plasmacytoid dendritic cells, which are associated with poor prognosis when infiltrate patients’ breast tumors. Two groups have identified BST2 isoforms of different lengths, generated by post-transcriptional modification. Based on these findings and our work on protein interaction of ILT7-BST2, we hypothesized that the expression of BST2 isoforms differed in normal and cancer tissues. To demonstrate that, we generated and characterized anti-long BST2 isoform monoclonal antibodies (mAbs). Results were compared with our previously developed mAb anti-BST2 full length molecule, 26F8, that recognizes a common epitope to all BST2 isoforms. By comparing the amino acid sequences of different isoforms of BST2, we identified a region of 24 residues on the extracellular domain of BST2, restricted to its long isoform. We then generated mAbs by hybridoma technology, immunizing BALB/c mice with the 24 mers peptide-KLH conjugated. Screening and positive selection of several clones were performed following our lab SOP. One of the mAbs, LA5, was selected based on positive ELISA against the immunogen and FACS staining of recombinant HEK293 cells expressing full-length BST2 protein. Pharmacokinetic determined using OCTET platform, revealed a KD of 4.61+/- 0.074 × 10−9 M for LA5. Epitope mapping of LA5 on BST2 overlapping peptides, confirmed specific recognition of full-length BST2 protein and the peptide used as immunogen. LA5 heavy and light chains were sequenced and analysis was performed by IgBlast and IMGTV. Binding of LA5 and 26F8 to the myeloma cell lines MM1 (B-lymphoblast) and U266 (B-lymphocyte) by flow cytometry, indicated 97-100% positive cells with both mAbs, with higher MFI values observed for clone 26F8: 5.8 and 1.8-fold-increase in MM1 and U266 cells respectively. IHC on formalin-fixed paraffin embedded tissues of human ductal breast carcinoma, revealed that LA5 staining was able to identify infiltrating carcinoma cells but not the normal surrounding acinar cells. In contrast, 26F8 staining did not discriminate between BST2+ malignant and surrounding normal acinar cells. Currently, we are analyzing a panel of &amp;gt;105 breast cancer tissue samples of different molecular classifications. Initial data revealed a high-level of expression of BST2 (by 26F8) on the epithelial area of the tumors, lower in the TME (tumor microenvironment-infiltrating immune cells) and almost negative expression in the surrounding fibroblasts rich stroma. Evaluation of IHC staining by LA5 is being processed and yet to be established. These IHC findings indicate a potential diagnostic value of LA5 to distinct between tumor and normal cells in patients’ specimens. Treatment prospects may arise during the process. Citation Format: Laura C. Bover, Ahmed Muhsin, Long T. Vien, Felipe Amaya Manzanares, Zhuang Wu, Janis D. Johnson, Diego Castro Reyes, Myrthala Moreno-Smith, Estefania Labanca, Irene Sorin, Silvia Saucedo, Silvina Gazzaniga, Alicia I. Bravo. Monoclonal antibody against BST2 isoform: potential tool for diagnostic and cancer therapy [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 6350.

Abstract 4801: Laurine acid enhances GEM-chemosensitivity targeting hypoxia-induced mitochondrial disfunction and stemness in pancreatic ductal adenocarcinoma

Abstract Hypoxia is a feature of the pancreatic ductal adenocarcinoma (PDA). Although gemcitabine (GEM) is widely used in chemotherapy for PDA, drug resistance restricts its clinical effectiveness. We have found that hypoxia contributes to chemoresistance, however the mechanism is still unclear. The aim of this study is to clarify the mechanism and to develop a new therapeutic strategy. First, we examined the effect of hypoxia to GEM sensitivity, stemness and energy metabolism in three human pancreatic ductal carcinoma cell lines. The inhibition rate by GEM was lower under CoCl2-induced chemical hypoxia than normoxia. Reactive oxygen species (ROS) after GEM was increased under normoxia but not under hypoxia. Flux analysis showed that both oxidative phosphorylation (OXPHOS) and glycolysis were suppressed under hypoxia, indicating a quiescent state. Next, we investigated the relationship between hypoxia and stemness. Hypoxia increased stem cell markers mRNA (C-Myc, Oct-3/4, CD24, CD44, CD133) expression and tumorsphere formation efficiency (TFE). In addition, hypoxia and GEM administration reduced sphere size but increased the number of spheres and TFE. This indicated that hypoxia and GEM suppressed proliferation, however promoted stemness. Second, to examine the mechanism of GEM resistance, we established two GEM-resistant cell lines from human PDA cells by continuous treatment with GEM and CoCl2-induced chemical hypoxia. One resistant cell line possessed reduced OXPHOS and decreased mitochondrial ROS levels, while the other resistant cell line possessed increased stemness. In both cell lines, ethidium bromide-stained mitochondrial DNA levels decreased, suggesting mitochondrial DNA damage. In addition, tetramethylrhodamine-stained mitochondrial membrane potential levels decreased, suggesting mitochondrial impairment. This indicated that long hypoxia and treatment with GEM induced mitochondrial impairment and acquisition of stemness, causing chemoresistance. We further investigated the effect of lauric acid (LAA), a medium-chain fatty acid, on GEM resistance. We have previously reported that LAA causes reprogramming of energy metabolism and induces cell death in cancer cells. Treatment with LAA restored GEM sensitivity in both GEM-resistant cell lines. Flux analysis showed that both OXPHOS and glycolysis were promoted, indicating displacement from quiescent state by LAA. These results suggest that decreased energy production, decreased mitochondrial ROS levels, and increased stemness associated with mitochondrial damage caused by GEM lead to GEM resistance, and that hypoxia may promote this process. Furthermore, forced activation of OXPHOS by LAA could be a tool to overcome GEM resistance. Citation Format: Tadataka Takagi, Hiroki Kuniyasu, Masayuki Sho, Rina Tani, Shiori Mori, Shingo Kishi, Yukiko Nishiguchi, Yudai Hojo. Laurine acid enhances GEM-chemosensitivity targeting hypoxia-induced mitochondrial disfunction and stemness in pancreatic ductal adenocarcinoma. [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 4801.

Abstract P6-14-08: Epithelial/stromal cross talks that induce malignant transition of human ductal carcinoma in situ

Abstract Background: A large fraction of human DCIS (&amp;gt;50%) may not need the multimodality treatment options currently offered to all patients. More importantly, while we may be overtreating many, we cannot identify those most at risk for invasion/metastasis. Revealing the cellular and molecular mechanisms by which some DCIS remain indolent while others advance to invasive and metastatic breast cancers is currently a clinical unmet need. Methods: To address this gap, we developed the Mouse-INtraDuctal (MIND) model, by which patient-derived (PDX) DCIS epithelial cells are injected intraductally and allowed to progress naturally in mice. Single cell RNA-sequencing (scRNA-seq) was utilized to profile the DCIS epithelial and stroma cells in progressors vs. non-progressors. To distinguish between stromal (diploid) cells and tumor (aneuploid) cells, we calculated Copy Number Aberration (CNA) profiles from RNA using CopyKAT. Cell-type specific differential gene expression analysis of DCIS epithelial cells and microenvironment cell types in progressors and non-progressors was performed. We also predicted putative ligand:receptor interactions between the tumor cells and cell types in the microenvironment by CellPhoneDB. Results: Among 37 PDX DCIS MIND models followed for a median of 9 months, 20 (54%) grafted into 95 glands, showed in vivo invasive progression (progressed) while 17 (46%), injected into 107 glands, remained non-invasive (non-progressed). ScRNA-seq was performed on 13 DCIS samples including 10 progressors and 3 non-progressors. Aneuploid cells were further analyzed to identify deferentially expressed genes that were upregulated in progressors compared to non-progressors (log2 fold=1, FDR p&amp;lt; 0.05).. Notable genes included NEAT1, EIF4EBP1, SCGB2A2, TFF1 and TFF3 that were upregulated in the progressors. NEAT1, the core structural component of the paraspeckles, is frequently overexpressed in human cancers and its expression is correlated with worse survival in cancer patients. NEAT1 drives tumor progression by regulating genes involved in cellular growth, migration, invasion, metastasis, EMT, stemness, radio- and chemoresistance, supporting its role as a potential biomarker and therapeutic target. TFF1/TFF3 mRNAs show increased expression in metastatic breast cancers. EIF4EBP1 is located on chrom 8p11-p12 which is frequently amplified in breast cancer and is associated with poor clinical prognosis. Further analysis using Cancer Hallmarks identified mitotic spindle, interferon signaling, DNA repair, oxidative phosphorylation and P53 pathway among the top signatures that were upregulated in the progressors. CellPhoneDB identified expression of several receptor/ligand interactions including CD74/MIF involved in epithelial/stromal and stromal/stromal cross talks that may play a role in DCIS invasive progression. Conclusions: Future studies will validate our findings using patient DCIS samples with known long-term outcome and in vivo MIND models to further refine risk associated biomarkers for invasion/metastasis and to identify more effective treatments. Citation Format: Aditi Rastogi, Fariba Behbod, Nicholas Navin, Jerome Lin, Linheng Li, Hua Li, Andrew K. Godwin, Alastair M. Thompson, Timothy Fields, Yan Hong. Epithelial/stromal cross talks that induce malignant transition of human ductal carcinoma in situ. [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 P6-14-08.

Growth and invasion inhibition of T47D ductal carcinoma cells by the association of docetaxel with a bioactive agent in neutral nanosuspension.

Introduction: The approach for drug delivery has impressively developed with the emergence of nanosuspension, particularly the targeted nanoemulsions (NEs). It can potentially improve the bioavailability of drugs, enhancing their therapeutic efficiency. This study aims to examine the potential role of NE as a delivery system for the combination of docetaxel (DTX), a microtubule-targeting agent, and thymoquinone (TQ) in the treatment of human ductal carcinoma cells T47D. Methods: NEs were synthesized by ultra-sonication and characterized physically by dynamic light scattering (DLS). A sulforhodamine B assay was performed to evaluate cytotoxicity, and a flow cytometry analysis for cell cycle, apoptosis, autophagy, and cancer stem cell evaluations. A quantitative polymerase chain reaction further assessed the epithelial-mesenchymal transition gene expirations of SNAIL-1, ZEB-1, and TWIST-1. Results: The optimal sizes of blank-NEs and NE-DTX+TQ were found at 117.3 ± 8 nm and 373 ± 6.8 nm, respectively. The synergistic effect of the NE-DTX+TQ formulation significantly inhibited the in vitro proliferation of T47D cells. It caused a significant increase in apoptosis, accompanied by the stimulation of autophagy. Moreover, this formulation arrested T47D cells at the G2/M phase, promoted the reduction of the breast cancer stem cell (BCSC) population, and repressed the expression of TWIST-1 and ZEB-1. Conclusion: Co-delivery of NE-DTX+TQ may probably inhibit the proliferation of T47D via the induction of apoptosis and autophagy pathways and impede the migration by reducing the BCSC population and downregulating TWIST-1 expression to decrease the epithelial-to-mesenchymal transition (EMT) of breast cancer cells. Therefore, the study suggests the NE-DTX+TQ formula as a potential approach to inhibit breast cancer growth and metastasis.

Immunohistochemical Expression of Cath-D as Prognostic Biological Marker and its Correlation with Clinical and Histopathological Parameters in Human Breast Cancer

Cathepsin D (Cath-D) is a soluble lysosomal aspartyl glycoprotease that can degrade the protein components of the matrix and free growth factors therein embedded, thus favoring tumor growth, invasion and angiogenesis. The aim of the present work was to investigate the expression of Cathepsin D as novel prognostic biomarker in human invasive ductal carcinoma (IDC) versus benign tumors and normal breast tissues as well as their correlation with different pathological and histological parameters. Immunohistochemical technique was used to examine the expression of Cath-D in normal, benign as well as in IDC. Present results showed higher expression of Cath-D in IDC comparing to normal and benign breast tissues.

Comparison of mammosphere formation from stem-like cells of normal breast, malignant primary breast tumors, and MCF-7 cell line

BackgroundMammosphere formation assay has become a versatile tool to quantify the activity of putative breast cancer stem cells in non-adherent in vitro cultures. However, optimizing the suspension culture system is crucial to establish mammosphere cultures from primary breast tumors.MethodsThis study aimed at determining the self-renewal and sphere-forming potential of breast cancer stem-like cells derived from human primary invasive ductal carcinoma and normal breast tissue samples, and MCF-7 breast cancer cell line using an optimal suspension culture system. Mammosphere-forming efficiency of the mammospheres generated from the tissue samples and cell line were compared. We evaluated the expression of CD44+/CD24−/low and CD49f+/EpCAM−/low phenotypes in the stem-like cells by flow cytometry. CK-18, CK-19, α-SMA, and EpCAM marker expression was assessed using immunohistochemical staining.ResultsBreast epithelial cells isolated from the three samples formed two-dimensional spheroids in suspension cultures. Interestingly, mammospheres formed from patient-derived primary breast tumors were enriched in breast cancer stem-like cells with the phenotype CD44+/CD24−/low and exhibited a relatively more number of large spheres when compared to the normal breast stem cells. MCF-7-derived SCs were more aggressive and resulted in the formation of a significantly higher number of spheroids. The expression of CK-18/CK-19 and α-SMA/EpCAM proteins was confirmed in breast cancer tissues.ConclusionsThus, the use of primary tumor specimens and breast cancer cell lines as suitable models for elucidating the breast cancer stem cell activity was validated using mammosphere culture system.

Immunohistochemical Expression of Fn14 and GP88 as Prognostic Biological Markers in Invasive Human Breast Cancer

Fibroblast growth factor-inducible 14 (Fn14), is a member of the tumor necrosis factor receptor super family that normally expressed in healthy tissues, but its expression is increased in injured tissue where it thought to play role in tissue remodeling. Progranulin or acrogranin is an 88-kDa glycoprotein identified by a biological screen for protein targets associated with high tumorigenicity. The aim of the present work was to investigate the expression of Fn14 and GP88 as novel prognostic biomarkers in human invasive ductal carcinoma (IDC) versus benign tumors and normal breast tissues as well as their correlation with different pathological and histological parameters. Immunohistochemical technique was used to examine the expression of Fn14 and GP88 in normal, benign as well as in IDC. Present results showed higher expression of Fn14 and GP88 in IDC comparing to normal and benign breast tissues.

Immunohistochemical Expression of Fn14 and Cath-D as Prognostic Biological Markers and Compare with Histological Reaction in Invasive Human Breast Cancer

Fibroblast growth factor-inducible-14 (Fn14) is a 14-kDa type I transmembrane receptor located on chromosome 16p13. Fn14 is a member of the tumor necrosis factor receptor super family that normally expressed in healthy tissues, but its expression is increased in injured tissue where it thought to play role in tissue remodeling. Cathepsin D (Cath-D) is a soluble lysosomal aspartyl glycoprotease that can degrade the protein components of the matrix and free growth factors therein embedded, thus favoring tumor growth, invasion and angiogenesis. The aim of the present work was to investigate the expression of Fn14 and Cathepsin D as novel prognostic biomarkers in human invasive ductal carcinoma (IDC) versus benign tumors and normal breast tissues as well as their correlation with different pathological and histological parameters. Immunohistochemical technique was used to examine the expression of Fn14 and Cath-D in normal, benign as well as in IDC. Present results showed higher expression of Fn14 and Cath-D in IDC comparing to normal and benign breast tissues.

Toxicity of new monophenolic synthetic activator of Keap1/Nrf2/ARE redox-sensitive signaling system &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt;

One of the promising areas of modern pharmacology is the development of «indirect antioxidants» capable of activating redox-sensitive signaling systems, primarily the Keap1/Nrf2/ARE system. Among its chemical inductors is the hydrophilic monosubstituted monophenol (3’-tert-butyl-4’-hydroxyphenyl)sodium propylthiosulfonate (TS-13) in development. The aim of the study was to investigate TS-13 antiproliferative activity against tumor cell line BT-474 in vitro and acute oral toxicity in mice in vivo. Material and methods. The relationship between TS-13 concentration and proliferative activity of human breast ductal carcinoma cell line BT-474 was determined using the MTT test, the IC&lt;sub&gt;50&lt;/sub&gt; was calculated and compared to the previously obtained for MCF-7 line; results were correlated with the functional properties of cells based on gene expression (in silico GSEA). In vivo acute toxicity was studied in 50 female C57Bl/6J mice, who received a TS-13 solution in distilled water at various doses by intragastric gavage. LD&lt;sub&gt;50&lt;/sub&gt; obtained experimentally and predicted in silico using the GUSAR web service were compared. Results and discussion. TS-13 inhibited the proliferation of BT-474 cells in a concentration-dependent manner (exponential approximation, IC&lt;sub&gt;50&lt;/sub&gt; = 59.5 μM) and was 2.2 times more toxic than for MCF-7 cells. This may be due to functional differences between the BT-474 and MCF-7 lines, as evidenced by the GSEA results. The LD&lt;sub&gt;50&lt;/sub&gt; value established in the in vivo experiment was 936 mg/kg body weight, the obtained value satisfactorily corresponds to the predicted in silico (561 mg/kg), although in reality the compound turned out to be somewhat less toxic than could be expected based on its structure. Conclusions. A study of the acute toxicity of the new water-soluble monophenol TC-13 allows the classification of it as slightly toxic (toxicity rating level 4) according to the Hodge – Sterner scale) or as moderately hazardous (hazard class 3) according to GOST 12.1.007-76.

Comparison of metabolite profiles and cytotoxicity of the black sea cucumber (Holothuria atra) dried with different drying techniques

Holothuria atra is a species of sea cucumber that could be a source of cytotoxic compounds. The present work aimed to determine the effect of different drying techniques on the profile of metabolites and cytotoxicity of sea cucumber extracts. Sea cucumbers were dried in the sun for 3 d, and then oven-dried at a temperature of 40, 50, and 60°C for 24 h. Fresh sea cucumbers without drying served as a control. The dried and fresh sea cucumbers were extracted by maceration for 12 h using ethanol. The metabolite profiling was performed using Fourier-transform infrared spectroscopy (FTIR) and high-performance liquid chromatography (HPLC) analyses, whereas the cytotoxicity was assayed by MTT method using the human breast ductal carcinoma cells (T47D). Analyses of FTIR and HPLC showed that the bioactive compounds of the ethanol extract were relatively stable during drying. This was in line with its cytotoxicity against T47D cells, which did not show significant differences between treatments. Based on these results, it was concluded that drying by sun or oven did not damage the bioactive compounds in the ethanolic extract of sea cucumber.

Design, synthesis and biological evaluation of a new series of arylidene indanones as small molecules for targeted therapy of non-small cell lung carcinoma and prostate cancer

In an attempt to identify small molecules for targeted therapy of non-small cell lung carcinoma (NSCLC) and prostate cancer (PCa), new arylidene indanones (1–10) were synthesized via the Claisen-Schmidt condensation of 5,6-methylenedioxy-1-indanone with p-substituted benzaldehyde. Compounds 1–10 were assessed for their cytotoxic effects on human lung adenocarcinoma (A549) and human pancreatic ductal carcinoma (PANC-1) cells as well as human normal lung fibroblast (CCD-19Lu) and human normal pancreatic ductal epithelial (hTERT-HPNE) cells. Among them, compounds 2, 4 and 10 were more effective on A549 and PANC-1 cells than cisplatin. Compounds 1 and 9 also showed more potent cytotoxic activity towards PANC-1 cells than cisplatin. In vitro assays were performed to assess their effects on DNA synthesis, apoptosis, caspase-3, mitochondrial membrane potential, intracellular calcium levels, morphological changes in cancer cells. Furthermore, all compounds were investigated for their inhibitory effects on cathepsin L (CatL) and cathepsin D (CatD). Compounds 2 and 4 exerted potent anti-NSCLC action through caspase-independent apoptosis induced by an increase in intracellular calcium level and correspondingly the disruption of the ΔΨm. These compounds also caused apoptotic morphological alterations in A549 cells. Compound 4 also inhibited both cathepsins but its inhibitory potency on CatL was more significant. Based on in vitro mechanistic assays, compound 4 was identified as a promising anticancer agent for targeted therapy of NSCLC. On the other hand, the marked anti- PCa activity of compound 1 mediated by apoptotic cell death is also noteworthy, but further enzymatic assays are required to elucidate its main mechanism of action.