4T1 Cells Research Articles

Abstract 4177: High-throughput in vivo monitoring of splenomegaly and tumor burden in a murine breast cancer model

Abstract Splenomegaly is a valuable parameter in mouse model cancer studies, as it can reflect the magnitude of cancer burden, hematopoietic activity, immune status, and can provide an indirect measure of cancer treatment efficacy. In liquid cancer models specifically (leukemia/lymphoma), spleen sizing is important as it is usually the only measure of in vivo tumor burden that can be assessed noninvasively without genetic labeling of tumor cells. Magnetic resonance imaging (MRI) and contrast-enhanced computed tomography (CT) are highly effective at evaluating spleen size in small animals. However, these techniques can impose limitations on large-cohort study designs due to high cost, long scan time, and ionizing radiation. Ultrasound is an alternative method with lower cost, high frame rate, and no ionization, but can suffer from poor inter-user variability, limited 2D field-of-view, and low throughput due to manual operation. In this work, we evaluated a novel 3D automated ultrasound scanner (Vega, Revvity, Inc.) for rapid assessment of splenomegaly in a murine cancer model. Female BALB/c mice (n=10) were implanted with 5e5 4T1 (ATCC) cells on the right flank. Mice were imaged at five timepoints over 25 days to track longitudinal progression of both spleen and tumor enlargement. Images were segmented manually in 3D using the “Slice Draw” and “Fill Between Slices” effects in SonoEQ v2.0.1 (Revvity, Inc.) to quantify tissue volume. After the final timepoint, mice were euthanized, and spleen weight was measured ex vivo. An additional cohort of age-matched mice without tumors (n=7) were imaged at the final timepoint to serve as controls. Imaging revealed a substantial enlargement of the spleen over time, from 30 ± 3 mm3 to 545 ± 105 mm3, representing an 18-fold increase. In the same time span, tumor volume grew to 1966 ± 87 mm3. Correlation analysis demonstrated good agreement between ultrasound volumes and ex vivo spleen weights (R² = 0.90). The results in this study demonstrate that noninvasive, nonlabelled quantification of murine spleen size with automated ultrasound is feasible, robust, and fast. The average time to acquire the spleen image for each mouse was less than 30 seconds. Future studies will apply this workflow in liquid cancer models, as well as explore using machine learning approaches to automatically segment spleen borders for faster data analysis. Citation Format: Craig McMannus, Tomek J. Czernuszewicz, Ryan C. Gessner, Jeffrey D. Peterson. High-throughput in vivo monitoring of splenomegaly and tumor burden in a murine breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4177.

Abstract 482: Advancing treatment for metastatic triple negative breast cancer: Liposomal oncolytic adenovirus as a novel neoadjuvant therapy

Abstract Introduction: Adenovirus type-5 (Ad) is considered a prominent viral vector in cancer therapy due to its modifiability for the selective lysis of cancer cells. However, challenges are posed in Ad delivery to cancers lacking coxsackievirus and adenovirus receptors (CARs). In response to this limitation, Ad-encapsulated DOTAP-folate liposomes were developed, which improved Ad delivery and heightened therapeutic efficacy through membrane fusion and folate receptor-mediated endocytosis. Methods: The 4T1 mammary carcinoma model, a CAR-negative triple-negative breast cancer (TNBC) cell line (ER-, PR-, HER2-), resistant to adenoviral infection, was utilized for studying the comparative in vitro transduction of green fluorescent protein (GFP)-expressing Ad (Ad-GFP). The study assessed Ad-GFP with and without DOTAP-folate liposomal encapsulation at a multiplicity of infection (MOI) of 50. Additionally, an oncolytic adenovirus, Ad-hTERT, with a human telomerase promoter, underwent liposomal encapsulation. CAR-negative 4T1 GFP-expressing cells and CAR-positive MDA-MB-231-GFP human breast adenocarcinoma TNBC cells were transduced in vitro at MOI 100 with Ad-hTERT, both with and without DOTAP-folate liposomal encapsulation. Results: CAR-negative 4T1 cells exhibited superior Ad-GFP transduction when encapsulated in DOTAP-folate liposomes, compared to the unencapsulated form (p < 0.001). For oncolytic adenovirus Ad-hTERT, encapsulation resulted in 100% cell lysis within 24 hours in 4T1-GFP cells, contrasting with 13% with unencapsulated Ad-hTERT (P < 0.0001). In CAR-positive MDA-MB-231-GFP TNBC cells, encapsulated Ad-hTERT showed enhanced efficacy, resulting in 100% cell lysis within 24 hours compared to 75% with unencapsulated Ad-hTERT (P < 0.001). Discussion: DOTAP-folate liposomal encapsulation enhances the efficacy of oncolytic Ad-hTERT in both CAR-negative and CAR-positive TNBC cells, establishing it as an optimal therapy for tumors with CAR heterogeneity. Ongoing in vivo studies with Balb/C immune-competent mice and the 4T1 tumor model will assess the effectiveness of encapsulation through intratumoral administration. This approach may extend the recurrence-free period post-surgical tumor removal. Citation Format: Jaimin R. Shah, Tao Dong, Abraham Phung, Sarah L. Blair, Omonigho Aisagbonhi, William C. Trogler, Andrew C. Kummel. Advancing treatment for metastatic triple negative breast cancer: Liposomal oncolytic adenovirus as a novel neoadjuvant therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 482.

Abstract 2680: Knockout on enhancer of zeste homolog 2 in triple-negative breast cancer model alters immune signaling and decreases tumor progression and lung metastasis

Abstract Triple-negative breast cancer (TNBC) is an aggressive and highly metastatic subtype of breast cancer. Lack of known targets, patient-to-patient variations of target antigens and resistance to immune checkpoint inhibitors make TNBC one of the most challenging cancers to treat. TNBC is enriched in tumor-infiltrating neutrophils (TINs). TINs are key contributors to pro-tumorigenic processes linked to “immunologically cold” tumors, in part by preventing recruitment of T-lymphocytes (TILs). Prior studies identified the Enhancer of zeste homolog 2 (EZH2) as a prominent methyltransferase expressed in primary and metastatic TNBC cells. EZH2 is an upstream regulator of the stimulator of interferon genes (STING), a critical innate sensor mediating anti-tumor responses that is inhibited in cold tumors. To date, EZH2/STING signaling in TINs is relatively unknown. Here, we hypothesized that EZH2 inhibition and subsequent STING activation could exert anti-tumor activity by modulating TINs. We derived EZH2-knockout (KO) and EZH2-overexpressed (OX) cell lines from the well-established 4T1 TNBC mouse cell line. We demonstrate a 10-fold decrease in lung metastasis and significant reduction of 4T1 EZH2 KO tumor growth compared to the parent 4T1 cell line. Concomitantly, the TIN:TIL ratio was significantly reduced in 4T1 EZH2 KO vs. control tumors. Overexpression of EZH2 in 4T1 TNBC cells did not affect the primary tumor growth or lung metastasis. A therapeutic study using the repeated injection of the STING agonist MSA-2 after tumor engraftment in vivo also demonstrated a decrease in lung metastasis and tumor growth in MSA-2 vs. vehicle injection with both 4T1 parent and EZH2 KO cell lines. Together, our findings suggest a key role for STING pathway modulation and TIN/TIL poise in primary TNBC progression and lung metastasis. Citation Format: Lenore Monterroza, Maria Parrilla, Sarah Samaranayake, Dormarie Rivera, Brian Dobosh, Periasamy Selvaraj, Rabindra M. Tirouvanziam. Knockout on enhancer of zeste homolog 2 in triple-negative breast cancer model alters immune signaling and decreases tumor progression and lung metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2680.

Abstract 708: Targeting aurora kinase B (AURKB) as a radiosensitizing strategy in syngeneic models of triple negative breast cancer (TNBC)

Abstract Purpose: Triple negative breast cancer (TNBC) is an aggressive breast cancer (BC) subtype with few treatment options. Radiation therapy (RT) is a mainstay therapy for the treatment of BC, but local recurrence following RT therapy is common. Consequently, decreasing local recurrence in patients with TNBC is a critical clinical need. Prior work demonstrated that AURKB is overexpressed in TNBC, and over-expression correlates with poor prognosis. Here, we examined the effects of AURKB inhibition as a novel radiosensitizing strategy in syngeneic TNBC models. Methods: Cell viability assays were used to determine the half-maximal inhibitory concentrations (IC50) of the AURKB inhibitors Barasertib-HQPA and SP-96 72 hours post treatment. Clonogenic survival assays were used to assess the radiosensitivity of TNBC murine cell lines to AURKB inhibition. In these assays, AURKB inhibitors were delivered at sub-IC50 concentrations 24 hours prior to RT, and radiation enhancement ratios (rERs) were calculated. Immunofluorescent microscopy using DAPI stain assessed micronuclei. Propidium iodide staining to assess aneuploidy was completed via flow cytometry. To assess the radiosensitizing effects of AURKB inhibition in vivo, the 4T1 syngeneic TNBC cell line was injected bilaterally into Balb/c mice and treated with Barasertib and RT. Tumor volume was recorded twice weekly throughout the study. Results: Aurora kinase B inhibitors (500-750 nM Barasertib-HQPA, 100-200 nM SP-96) delivered 24 hours prior to radiation therapy induced radiosensitization in the murine TNBC cells 4T1 (rER: 1.24-1.56) and Py8119 (rER: 1.51-1.72). Mechanistically, combined AURKB inhibition and RT significantly increased micronuclei formation in 4T1 cells 24 hours after RT compared to vehicle control (p<0.0001). Furthermore, combined AURKB inhibition and RT induced aneuploidy in murine TNBC cell lines 24 hours after radiation therapy compared to vehicle control. Combined AURKB inhibition (Barasertib 25 mg/kg, IP) and RT (8 Gy RT in 1 fraction) significantly decreased tumor volume compared to mice that had received vehicle control (777 ± 61 mm3 vs 1623 ± 126 mm3; p <0.0001). Conclusion: AURKB inhibition induces radiosensitization in syngeneic models of TNBC and leads to increased micronuclei and aneuploidy, suggesting a mechanism of sensitization. These results suggest that AURKB is a potential radiosensitizing strategy for the treatment of triple negative disease. Ongoing studies are further refining the underlying mechanisms of AURKB inhibition and RT on the antitumoral immune response. Citation Format: Kassidy M. Jungles, Zhuwen Wang, Caroline Bishop, Cydnee Wilson, Meilan Liu, Jadyn James, Michael Green, James M. Rae, Corey W. Speers, Lori J. Pierce. Targeting aurora kinase B (AURKB) as a radiosensitizing strategy in syngeneic models of triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 708.

Abstract 3076: Unveiling the therapeutic potential of β-catenin inhibition in triple negative breast cancers: Targeting the Warburg effect

Abstract Introduction: Triple Negative Breast Cancer (TNBC) exhibits increased glycolysis, known as the Warburg effect. Recent research, including our own Nanostring nCounter studies, identify β-catenin as an oncoprotein linked to elevated glycolysis in TNBC tumors. Our current research explores the impact of β-catenin inhibitors on glycolysis in TNBC cell lines and an in vivo CDX model. Method: We employed immunohistochemistry to assess 98 TNBCs for the presence of β-catenin, PFKP, LDH-A, and MCT1 proteins, exploring their correlation with clinicopathological factors. In in-vitro experiments using MDA-MB-231, MDA-MB-468, and 4T1 cell lines, we investigated how β-catenin inhibitors (XAV939 & Axitinib) affected aerobic glycolysis components, and TNBC cell characteristics (proliferation, migration, invasion, and stemness). For in-vivo validation, 4T1 TNBC xenografts were employed to confirm our findings. Results: Among the 98 TNBC patients, 63 (64%) displayed cytoplasmic/or nuclear β-catenin staining. Additionally, cytoplasmic accumulation of PFKP was observed in 52/98 (53%), increased cytoplasmic LDH-A expression in 57/98 (58%), and membranous MCT-1 expression in 50/98 (51%) TNBC patients. Notably, significant positive correlations were found between nuclear/cytoplasmic β-catenin and cytoplasmic PFKP (p = 0.001) as well as membranous MCT1 (p = 0.014). Furthermore, β-catenin, PFKP, LDHA, and MCT1 proteins were overexpressed in all TNBC cell lines thereby supporting their glycolytic nature. Comparative analyses between XAV939 and Axitinib revealed that Axitinib significantly reduced various TNBC cell parameters, including migration, invasion, lactate production, colony-forming ability, and expression of aerobic glycolytic pathway proteins. In the in-vivo studies, 30mg/kg Axitinib in a female Balb/c-4T1 xenograft model resulted in a substantial reduction in tumor volume and weight. Conclusion: Our findings show that increased levels of β-catenin play an important role in regulating aerobic glycolysis in TNBCs. Furthermore, our findings also highlight Axitinib's potential as an effective therapeutic agent for the treatment of β-catenin-positive TNBCs. Citation Format: Sheikh Mohammad Umar, Shruti kahol, Arundhathi Dev JR, Akanksha Kashyap, Sandeep R. Mathur, Ajay Gogia, S.V.S Deo, Chandra Prasad. Unveiling the therapeutic potential of β-catenin inhibition in triple negative breast cancers: Targeting the Warburg effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3076.

Abstract 4219: Establishing CAF-like and TAM-like transformation induced by TNBC culture supernatant in 3D in-vitro culture as a model for targeted drug testing

Abstract Several previous studies have highlighted the significance of cell-to-cell communication, or crosstalk, between tumor cells and non-tumorigenic cells in the context of cancer progression and metastasis. Within the tumor microenvironment (TME), these interactions have the potential to alter the phenotypes and behaviors of normal cells. The use of 2D in-vitro cultures has been limited due to their inability to accurately replicate the intricate in-vivo TME. Conditioned medium (CM) derived from cultured cancer cells contains secreted factors that may influence the phenotype and functionality of normal cells. In our investigation, the exposure of normal murine fibroblast NIH3T3 and macrophage RAW 264.7 cells to conditioned medium (CM) obtained from malignant mammary epithelial 4T1 cells (4T1CM) resulted in a modified phenotype with enhanced cell viability. Treatment with 4T1CM led to the upregulation of genes, including α-smooth muscle actin (αSMA), IL-10, CD206, and vascular endothelial growth factor (VEGF), in NIH3T3 and RAW 264.7 cells compared to their respective control cells. Additionally, NIH3T3 cells treated with 4T1CM exhibited an epithelial-mesenchymal transition (EMT) phenotype, as indicated by the regulation of EMT markers such as E-cadherin, β-catenin, N-cadherin, and Vimentin. Notably, RAW 264.7 cells treated with 4T1CM showed an upregulation of cyclooxygenase-2 (COX-2) and programmed death-ligand 1 (PDL-1), suggesting a propensity for an inhibitory immune response. Moreover, NIH3T3 cells conditioned with 4T1CM demonstrated an upregulation of stemness markers, including sex determining region Y-box 2 and Aldehyde dehydrogenase. In summary, our study highlights the potential role of 4T1CM in transforming normal NIH3T3 and RAW 264.7 cells into cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). Ongoing proteomics analysis aims to map differentially expressed proteins potentially regulating the 4T1CM-induced transformation. To further investigate, a series of experiments using mono- and co-cultures of these cells are underway, specifically targeting the transformed macrophages and fibroblasts with the aim of either re-educating or eliminating them. This treatment involves drug combinations that specifically target the identified signaling pathway intermediates, offering novel therapeutic intervention strategies. Citation Format: Biplov Sapkota, Naveen Chintalaramulu, Abhishek Pandit, Shilpa Thota, Rizwana Begum, Amirsalar Mansouri, Jiri Adamec, Joseph Francis. Establishing CAF-like and TAM-like transformation induced by TNBC culture supernatant in 3D in-vitro culture as a model for targeted drug testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4219.

Abstract 6850: Unraveling molecular factors contributing to metallomic dyshomeostasis induced by triple-negative breast cancer using single-cell RNA sequencing

Abstract Triple-negative breast cancer (TNBC) comprises 20% of breast cancer cases and exhibits an aggressive phenotype with increased metastasis; these patients have limited treatment options, poorer prognosis and higher mortality than other breast cancers (BC). Metallomics is a relatively new discipline that studies the abundance of metal ions in biological samples. Metallomic analysis has identified differences in BC patients in elements such as iron (Fe) and zinc (Zn) but it is unclear if these reflect metabolic changes in the tumor. In this study, we conducted metallomic profiling to characterize 55 trace elements in the circulation and in the tumor microenvironment of orthotopic mammary fat pad tumors from two isogenic BC cell lines (highly metastatic 4T1; nonmetastatic 67NR). We complemented this analysis with single-cell RNA sequencing of the tumor to determine if complementary changes occur in molecules known to utilize metals as co-factors or transporters and identify drivers of metal metabolism in different tumors. We performed analysis on systemic metal elements by examining whole blood (including blood cells and platelets) and plasma separately. Tumor samples were assessed at the whole tissue level, and we also profiled separated immune or non-immune cells from the tumor. Li, Na, Mg, P, S, K, Ca, Fe, Cu, Zn, Rb were detectable within 100ul of input of whole blood or plasma. Metallomic analysis of the tumor and associated cell populations detected differential levels of Na, Mg, P, S, K Ca, Fe, Zn, and Rb from an average of 200mg of tissue or 2 million cells. Based on scRNAseq analysis, 67NR tumors were composed of greater proportions of cancer cells relative to the 4T1 tumors (79.1% vs 58.8% of tumor cells respectively) and endothelial cells (2.5% vs 1.8%). 4T1 tumors contained an increased abundance of cancer-associated fibroblasts (5.6% vs 2.1%) as well as immune infiltration (33.8% vs 16.3%). Fibroblasts and macrophage populations also showed elevated metal transporter expression levels based on gene expression relative to other tumor residing cell types yet minor differences induced by cancer type. Cancer cells express high levels of metal transport genes (Zn, Ca, Co, Na, Mg, Fe). 67NR cancer cells exhibited increased gene expression of many metal-transporting proteins including numerous solute carrier (SLC) group of membrane transport proteins such as Slc41a2 when compared to the more aggressive 4T1 cells. This study underscores the potential of linking metallomic profiling to tumor composition and cancer behaviors, offering a pathway for identifying novel biomarkers to differentiate aggressive and non-aggressive forms of breast cancer. This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. Citation Format: Nicholas R. Hum, John M. Rolison, Nicole F. Leon, Ali Navid, Aimy Sebastian, Josh Wimpenny, Gabriela Loots. Unraveling molecular factors contributing to metallomic dyshomeostasis induced by triple-negative breast cancer using single-cell RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6850.

Abstract 6029: 225Ac/89Zr labeled anti nectin 4 radioimmunoconjugates as theranostics against nectin 4 positive triple negative breast cancer

Abstract Nectin4 (N4) is a biomarker that is overexpressed in ~62% of triple negative breast cancer (TNBC) and its lack of expression in normal tissues makes it an ideal target for the therapy and PET imaging of TNBC. Methods: We developed a fully human antibody against nectin 4 (anti N4) using phage display. Anti N4 was radiolabeled with 89Zr & 225Ac, respectively, for imaging & radiotherapy using TNBC xenograft & syngeneic mouse models. TNBC murine 4T1 cells were transduced with human N4 and N4 expression was confirmed by FACS. Biodistribution & PET imaging of 89Zr anti N4 radioimmunoconjugate (RIC) was studied in mice bearing N4 positive xenografts. Dosimetry of 225Ac anti N4 was studied in healthy mice & the therapeutic efficacy was evaluated for 90 days in mice bearing MDA MB 468 xenograft & transduced 4T1 syngeneic models. Mice were treated with 2 doses of 350 nCi administered at 10 days apart. Tumor growth was monitored by a digital caliper. Results: The pharmacokinetic profile of 89Zr-anti-N4 RIC showed biphasic distribution with a moderate elimination of 63 h. PET imaging & biodistribution of 89Zr anti N4 in mice bearing MDA MB 468 xenograft showed high tumor uptake of 13.2 ± 1.12 %IA/g at 120 h. 225Ac anti N4 was effectively internalized in MDA MB 468 and was cytotoxic to the cells with IC50 of 1.2 kBq/mL. Treatment with 225Ac anti N4 led to complete tumor remission in all mice bearing MDA MB 468 xenografts and 4T1 syngeneic model by day 20 and 28, respectively. Conclusions: 89Zr anti N4 was an effective PET imaging probe with specific uptake in tumor. 225Ac anti N4 was effective as a therapeutic agent resulting in complete remission of tumors in both mouse xenograft & syngeneic models. These results are promising for the development of anti-N4 RICs as theranostics for TNBC. Citation Format: Hanan Babeker, Fabrice Njotu, Jessica Ketchmen, Florence Tikum, Alireza Doroudi, Emmanuel Nwangele, Maruti Uppalapati, Humphrey Fonge. 225Ac/89Zr labeled anti nectin 4 radioimmunoconjugates as theranostics against nectin 4 positive triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6029.

Abstract 7311: Lipid soluble vitamin B1 derivatives, benfotiamine and fursultiamine, prevent breast cancer cells growth

Abstract Breast cancer is the leading cause of cancer death in women in the US and the second leading cancer after skin cancer in women worldwide. According to the CDC, women from 45-84 years of age are most at risk, with approximately 30% of deaths in this group being from cancer. Increased free radical formation, and oxidative and inflammatory responses have been shown to be major contributing factors for the progression of breast cancer. Several antioxidants and vitamins have been tested for their efficacy in preventing breast cancer. However, the role of vitamin B1 derivatives in the prevention of breast cancer is not known. Therefore, we hypothesize that with potent anti-oxidative and anti-inflammatory actions, lipid-soluble derivatives of vitamin B1 (thiamine), such as benfotiamine and fursultiamine, could prevent breast cancer growth and spread. We have treated human breast cancer cells such as MCF-7 and T-4D1 and mouse breast cancer cells 4T1 with vitamin B1 derivatives and examined the cancer cells growth. Our results indicate that treatment of various breast cancer cells with benfotiamine and fursultiamine prevents the proliferation of cancer cells in a dose-dependent manner. Further, both benfotiamine and fursultiamine by regulating the activation of caspase-3 and cleavage of PARP protein could prevent breast cancer cells growth. Benfotiamine and fursultiamine also regulates generation of reactive oxygen species in breast cancer cells. Further, vitamin B1 derivatives regulate the expression of various anti-apoptotic, pro-apoptotic, survival and inflammatory factors in both human and mouse breast cancer cells. We next planned to examine the effect of vitamin B1 derivatives on breast cancer growth in vivo using athymic nude mouse models. In conclusion, our in vitro results suggest that vitamin B1 derivatives, benfotiamine and fursultiamine, by promoting the apoptotic pathways prevent breast cancer cells growth and could be further developed as chemopreventive drugs. Citation Format: Caroline Ciaciuch O'Neill, Aveline P. Langmead, Kota V. Ramana. Lipid soluble vitamin B1 derivatives, benfotiamine and fursultiamine, prevent breast cancer cells growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7311.

ROS mediated Cu[Fe(CN)5NO] nanoparticles for triple negative breast cancer: A detailed study in preclinical mouse model

Triple negative breast cancer (TNBC) is an aggressive form of tumor, more prevalent in younger women resulting in poor survival rate (2nd in cancer deaths) because of its asymptomatic existence. The most popular and convenient approach for the treatment of TNBC is chemotherapy which is associated with several limitations. Considering the importance of nanotechnology in health care system, in the present manuscript, we have designed and developed a simple, efficient, cost effective, and ecofriendly method for the synthesis of copper nitroprusside analogue nanoparticles (Cu[Fe(CN)5NO] which is abbreviated as CuNPANP that may be the potential anti-cancer nanomedicine for the treatment of TNBC. Copper (present in CuNPANP) is used because of its affordability, nutritional value and various biomedical applications. The CuNPANP are thoroughly characterized using several analytical techniques. The in vitro cell viability (in normal cells) and the ex vivo hemolysis assay reveal the biocompatible nature of CuNPANP. The anti-cancer activity of the CuNPANP is established in TNBC cells (MDA-MB-231 and 4T1) through several in vitro assays along with plausible mechanisms. The intraperitoneal administration of CuNPANP in orthotopic breast tumor model by transplanting 4T1 cells into the mammary fat pad of BALB/c mouse significantly inhibits the growth of breast carcinoma as well as increases the survival time of tumor-bearing mice. These results altogether potentiate the anti-cancer efficacy of CuNPANP as a smart therapeutic nanomedicine for treating TNBC in near future after bio-safety evaluation in large animals.

Facile synthesis and polymerization of 1,4,5-oxadithiepan-2-one for disulfide-based redox-responsive drug delivery

Chemotherapeutic drugs have been shown to be promising for the treatment of tumors, however, they are associated with several disadvantages including low aqueous solubility, non-specificity, and poor plasma stability. Hence, they accumulate in the health organs and create several adverse effects. Drug encapsulated stimuli-responsive nanoparticles bearing redox-responsive disulfide linkages have emerged as promising delivery vehicle to address these limitations. A glutathione (GSH) responsive disulfide-based drug delivery system based on a polyethylene glycol methyl ether (PEGME) and a polydisulfide (PDS) diblock copolymer has been developed. The cyclic lactone containing a disulfide link, 1,4,5-oxadithiepan-2-one, was prepared using a thiol-free method utilizing the reaction of 2-bromoethyl bromoacetate with sodium disulfide. Ring-opening polymerization (ROP) of 1,4,5-oxadithiepan-2-one using 1-butanol in the presence of Sn(oct)2 generated the homopolymer PDS whereas the diblock copolymer PEGME-b-PDS was obtained when PEGME was used as a macroinitiator. In aqueous solution, PEGME-b-PDS self-assembled into spherical-shaped micelles with a hydrodynamic radius of 146 and 116 nm as measured by Field-emission Scanning Electron Microscopy (FESEM) and Dynamic Light Scattering (DLS), respectively. At pH = 7.4, a Doxorubicin (DOX) loaded PEGME-b-PDS micelle showed a cumulative release of ∼98.2, ∼9.9, and ∼1.9 % in presence of 2 mM, 2 μM DTT and in absence of DTT, respectively clearly demonstrating the redox responsiveness of the PDS block. The best fit of kinetic data using with the Korsmeyer-Peppas model suggests a Fickian diffusion as well as chain-relaxation controlled drug release process. DOX loaded micelles and free DOX displayed similar cytotoxicity towards both 4T1 and MCF-7 cells indicating that the encapsulation inside the micelle did not affect the anticancer efficacy of the drug. The drug loaded micelle showed higher cellular uptake than free DOX in both 4T1 and MCF-7 lines and lower cellular uptake than free DOX in 3T3-L1 cell lines. This polymeric micellar nanoparticle may be further exploited for encapsulation of other hydrophobic drugs.

Seven glycosides from the roots of Scrophularia ningpoensis

Three new glycosides (1–3) together with four known ones (4–7) were isolated from the roots of Scrophularia ningpoensis. Their structures were elucidated through extensive analysis of HR-ESI-MS, NMR, and ECD spectra. The anti-inflammatory potential of the new compounds was evaluated by measuring their ability to inhibit lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW 264.7 macrophage cells. In addition, the cytotoxic effects of the new compounds on HepG2, 4T1, and A549 cell lines were evaluated.

Trimethyltin(IV) Bearing 3-(4-Methyl-2-oxoquinolin-1(2H)-yl)propanoate Causes Lipid Peroxidation-Mediated Autophagic Cell Death in Human Melanoma A375 Cells.

A novel trimethyltin(IV) complex (Me3SnL), derived from 3-(4-methyl-2-oxoquinolin-1(2H)-yl)propanoate ligand, has been synthesized and characterized by elemental microanalysis, UV/Vis spectrophotometry, FT-IR and multinuclear (1H, 13C and 119Sn) NMR spectroscopies. Furthermore, the structure of the ligand precursor HL was solved using SC-XRD (single-crystal X-ray diffraction). The prediction of UV/Vis and NMR spectra by quantum-chemical methods was performed and compared to experimental findings. The protein binding affinity of Me3SnL towards BSA was determined by spectrofluorometric titration and subsequent molecular docking simulations. Me3SnL has been evaluated for its in vitro anticancer activity against three human cell lines, MCF-7 (breast adenocarcinoma), A375 (melanoma) and HCT116 (colorectal carcinoma), and three mouse tumor cell lines, 4T1 (breast carcinoma), B16 (melanoma) and CT26 (colon carcinoma), using MTT and CV assays. The strong inhibition of A375 cell proliferation, ROS/RNS upregulation and robust lipid peroxidation lead to autophagic cell death upon treatment with Me3SnL.

Cyclin-dependent kinase inhibitors enhance programmed cell death protein 1 immune checkpoint blockade efficacy in triple-negative breast cancer by affecting the immune microenvironment.

Clinical studies on programmed death-ligand 1 (PD-L1) immune checkpoint inhibitors for treating triple-negative breast cancer (TNBC) have shown unsatisfactory efficacy due to low tumor-infiltrating lymphocyte (TIL) levels. Inhibitors targeting cyclin-dependent kinase (CDK) proteins can affect the immune microenvironment, increase TIL levels, and promote antitumor immunity, thus providing a new direction for TNBC treatment strategies. The authors tested three CDK inhibitors on the TNBC cell lines MDA-MB-231 and 4T1 and validated their antitumor effects and impact on the immune microenvironment using multiple detection methods. They verified the efficacy and immune-related mechanisms of different combination therapy experiments in a 4T1 cell-transplanted BALB/c mouse model. Treatment with CDK inhibitors for 72 hours inhibited cell proliferation, clone formation, migration, and cell-cycle arrest and induced apoptosis in human breast cancer MDA-MB-231 cells and mouse breast cancer 4T1 cells. CDK inhibitors suppressed DNA methylation by downregulating DNMT1, DNMT3a, and DNMT3b expression. These three inhibitors promoted the secretion of various chemokines, enhanced tumor cell antigen presentation, and increased PD-L1 expression. CDK inhibitors improved the efficacy of immunotherapy in animal models and increased TIL levels. Combination therapy with CDK and PD-L1 immune checkpoint inhibitors affects the immune microenvironment, promotes antitumor immunity, and improves the efficacy of immunotherapy for TNBC.

Accumulation and toxicity of biologically produced gold nanoparticles in different types of specialized mammalian cells.

The biologically produced gold nanoparticles (AuNPs) are novel carriers with promising use in targeted tumor therapy. Still, there are no studies regarding the efficacy of nanoparticle internalization by cancer and noncancer cells. In this study, AuNPs were produced by Fusarium oxysporum and analyzed by spectrophotometry, transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDS), and Zetasizer. Obtained AuNPs were about 15nm in size with a zeta potential of -35.8mV. The AuNPs were added to cancer cells (4T1), noncancer cells (NIH/3T3), and macrophages (RAW264.7). The viability decreased in 4T1 (77 ± 3.74%) in contrast to NIH/3T3 and RAW264.7 cells (89 ± 4.9% and 90 ± 3.5%, respectively). The 4T1 cancer cells also showed the highest uptake and accumulation of Au (∼80% of AuNPs was internalized) as determined by graphite furnace atomic absorption spectroscopy. The lowest amount of AuNPs was internalized by the NIH/3T3 cells (∼30%). The NIH/3T3 cells exhibited prominent reorganization of F-actin filaments as examined by confocal microscopy. In RAW264.7, we analyzed the release of proinflammatory cytokines by flow cytometry and we found the AuNP interaction triggered transient secretion of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). In summary, we proved the biologically produced AuNPs entered all the tested cell types and triggered cell-specific responses. High AuNP uptake by tumor cells was related to decreased cell viability, while low nanoparticle uptake by fibroblasts triggered F-actin reorganization without remarkable toxicity. Thus, the biologically produced AuNPs hold promising potential as cancer drug carriers and likely require proper surface functionalization to shield phagocytizing cells.

Exosomes Derived from Mouse Breast Carcinoma Cells Facilitate Diabetic Wound Healing.

Exosomes derived from breast cancer have been reported to play a role in promoting cell proliferation, migration, and angiogenesis, which has the potential to accelerate the healing process of diabetic wounds. The aim of this investigation was to examine the function of exosomes originating from 4T1 mouse breast carcinoma cells (TEXs) in the process of diabetic wound healing. The assessment of primary mouse skin fibroblastscell proliferation and migration was conducted through the utilization of CCK-8 and wound healing assays, while the tube formation of HUVECs was evaluated by tube formation assay. High-throughput sequencing, RT-qPCR and cell experimentswere used to detect the roles ofmiR-126a-3pin HUVECsfunctions in vitro. The in vivo study employed a model of full-thickness excisional wounds in diabetic subjects to explore the potential therapeutic benefits of TEXs. Immunohistochemical and immunofluorescent techniques were utilized to evaluate histological changes in skin tissues. The findings suggested that TEXs facilitate diabetic wound healing through the activation of cell migration, proliferation, and angiogenesis. An upregulation of miR-126a-3p has been observed in TEXs, and it has demonstrated efficient transferability from 4T1 cells to HUVEC cells. The activation of the PI3K/Akt pathway has been attributed to miR-126a-3p derived from TEXs. The promotion of chronic wound healing can be facilitated by TEXs through the activation of cellular migration, proliferation, and angiogenesis. The activation of the PI3K/Akt pathway by miR-126a-3p originating from TEXs has been discovered, indicating a potential avenue for enhancing the regenerative capabilities of wounds treated with TEXs.

Epiberberine inhibits bone metastatic breast cancer-induced osteolysis

Ethnopharmacological relevanceThe anti-tumor related diseases of Coptidis Rhizoma (Huanglian) were correlated with its traditional use of removing damp-heat, clearing internal fire, and counteracting toxicity. In the recent years, Coptidis Rhizoma and its components have drawn extensive attention toward their anti-tumor related diseases. Besides, Coptidis Rhizoma is traditionally used as an anti-inflammatory herb. Epiberberine (EPI) is a significant alkaloid isolated from Coptidis Rhizoma, and exhibits multiple pharmacological activities including anti-inflammatory. However, the effect of epiberberine on breast cancer and the inflammatory factors of metastatic breast cancer-induced osteolysis has not been demonstrated clearly. Aim of the studyBone metastatic breast cancer can lead to osteolysis via inflammatory factors-induced osteoclast differentiation and function. In this study, we try to analyze the effect of epiberberine on breast cancer and the inflammatory factors of metastatic breast cancer-induced osteolysis. MethodsTo evaluate whether epiberberine could suppress bone metastatic breast cancer-induced osteolytic damage, healthy female Balb/c mice were intratibially injected with murine triple-negative breast cancer 4T1 cells. Then, we examined the inhibitory effect and underlying mechanism of epiberberine on breast cancer-induced osteoclastogenesis in vitro. Xenograft assay was used to study the effect of epiberberine on breast cancer cells in vivo. Moreover, we also studied the inhibitory effects and underlying mechanisms of epiberberine on RANKL-induced osteoclast differentiation and function in vitro. ResultsThe results show that epiberberine displayed potential therapeutic effects on breast cancer-induced osteolytic damage. Besides, our results show that epiberberine inhibited breast cancer cells-induced osteoclast differentiation and function by inhibiting secreted inflammatory cytokines such as IL-8. Importantly, we found that epiberberine directly inhibited RANKL-induced differentiation and function of osteoclast without cytotoxicity. Mechanistically, epiberberine inhibited RANKL-induced osteoclastogensis via Akt/c-Fos signaling pathway. Furthermore, epiberberine combined with docetaxel effectively protected against bone loss induced by metastatic breast cancer cells. ConclusionsOur findings suggested that epiberberine may be a promising natural compound for treating bone metastatic breast cancer-induced osteolytic damage by inhibiting IL-8 and is worthy of further exploration in preclinical and clinical trials.

Rujifang inhibits triple-negative breast cancer growth via the PI3K/AKT pathway

Ethnopharmacological relevanceRujifang (RJF) constitutes a traditional Chinese medicinal compound extensively employed in the management of triple-negative breast cancer (TNBC). However, information regarding its potential active ingredients, antitumor effects, safety, and mechanism of action remains unreported. Aim of the studyTo investigate the efficacy and safety of RJF in the context of TNBC. Materials and methodsWe employed the ultra high-performance liquid chromatography-electrospray four-pole time-of-flight mass spectrometry technique (UPLC/Q-TOF-MS/MS) to scrutinize the chemical constituents of RJF. Subcutaneously transplanted tumor models were utilized to assess the impact of RJF on TNBC in vivo. Thirty female BLAB/c mice were randomly divided into five groups: the model group, cyclophosphamide group, and RJF high-dose, medium-dose, and low-dose groups. A total of 1 × 106 4T1 cells were subcutaneously injected into the right shoulder of mice, and they were administered treatments for a span of 28 days. We conducted evaluations on blood parameters, encompassing white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), platelet count (PLT), neutrophils, lymphocytes, and monocytes, as well as hepatorenal indicators including alkaline phosphatase (ALP), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), albumin, and creatinine (CRE) to gauge the safety of RJF. Ki67 and TUNEL were detected via immunohistochemistry and immunofluorescence, respectively. We prepared RJF drug-containing serum for TNBC cell lines and assessed the in vitro inhibitory effect of RJF on tumor cell growth through the CCK8 assay and cell cycle analysis. RT-PCR was employed to detect the mRNA expression of cyclin-dependent kinase and cyclin-dependent kinase inhibitors in tumor tissues, and Western blot was carried out to ascertain the expression of cyclin and pathway-related proteins. Results100 compounds were identified in RJF, which consisted of 3 flavonoids, 24 glycosides, 18 alkaloids, 3 amino acids, 8 phenylpropanoids, 6 terpenes, 20 organic acids, and 18 other compounds. In animal experiments, both CTX and RJF exhibited substantial antitumor effects. RJF led to an increase in the number of neutrophils in peripheral blood, with no significant impact on other hematological indices. In contrast, CTX reduced red blood cell count, hemoglobin levels, and white blood cell count, while increasing platelet count. RJF exhibited no discernible influence on hepatorenal function, whereas Cyclophosphamide (CTX) decreased ALP, GOT, and GPT levels. Both CTX and RJF reduced the expression of Ki67 and heightened the occurrence of apoptosis in tumor tissue. RJF drug-containing serum hindered the viability of 4T1 and MD-MBA-231 cells in a time and concentration-dependent manner. In cell cycle experiments, RJF diminished the proportion of G2 phase cells and arrested the cell cycle at the S phase. RT-PCR analysis indicated that RJF down-regulated the mRNA expression of CDK2 and CDK4, while up-regulating that of P21 and P27 in tumor tissue. The trends in CDKs and CDKIs protein expression mirrored those of mRNA expression. Moreover, the PI3K/AKT pathway displayed downregulation in the tumor tissue of mice treated with RJF. ConclusionRJF demonstrates effectiveness and safety in the context of TNBC. It exerts anti-tumor effects by arresting the cell cycle at the S phase through the PI3K-AKT pathway.

Assessing Radiation Effects on Chemo-Treated BT20 and 4T1 Breast Cancer, and Neuroblastoma Cell Lines: A Study of Single and Multiple-Cell Ionization via Infrared Laser Trapping

Background: Our study aimed to assess the radiation sensitivity of BT20, a human breast tumor cell line, using the laser-trapping technique and compare it with N2a and 4T1 cells. Additionally, we investigated the impact of the antitumor compound 2-Dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD) on radiation sensitivity. Methods and Materials: We employed laser trapping to calculate both the threshold ionization energy (TIE) and threshold radiation dose (TRD) for BT20, N2a, and 4T1 cells. We assessed the effect of DMDD on BT20 cells’ radiosensitivity and conducted comparisons across these cell lines. Results: Our findings reveal that DMDD significantly enhances the radiosensitivity of BT20 breast carcinoma cells. Moreover, we observed distinct trends in TIE and TRD across the three cell lines, with differences attributed to variations in cell size and composition. When multiple cell ionizations were considered, a notable reduction in TRD was observed, implicating factors such as the chain effect of ionizing radiation and the influence of DMDD. The study found that TIE increased with the number of cells in the trap while TRD consistently decreased across all three cell lines, suggesting comparable radiation sensitivity, and oligostilbene treatment further reduced TRD, presenting the potential for enhancing therapeutic ratios in cancer treatment. Conclusion: The antitumor compound DMDD enhances the radiosensitivity of BT20 breast carcinoma cells, highlighting its potential in cancer treatment. Furthermore, our study underscores the impact of cell size and multiple-cell ionizations on TRD. Leveraging laser trapping techniques, biocompatible nanoparticles, and advanced optical tweezers opens promising avenues for personalized and effective cancer therapy approaches.

Modulated Electro-Hyperthermia Accelerates Tumor Delivery and Improves Anticancer Activity of Doxorubicin Encapsulated in Lyso-Thermosensitive Liposomes in 4T1-Tumor-Bearing Mice.

Modulated electro-hyperthermia (mEHT) is an adjuvant cancer therapy that enables tumor-selective heating (+2.5 °C). In this study, we investigated whether mEHT accelerates the tumor-specific delivery of doxorubicin (DOX) from lyso-thermosensitive liposomal doxorubicin (LTLD) and improves its anticancer efficacy in mice bearing a triple-negative breast cancer cell line (4T1). The 4T1 cells were orthotopically injected into Balb/C mice, and mEHT was performed on days 9, 12, and 15 after the implantation. DOX, LTLD, or PEGylated liposomal DOX (PLD) were administered for comparison. The tumor size and DOX accumulation in the tumor were measured. The cleaved caspase-3 (cC3) and cell proliferation were evaluated by cC3 or Ki67 immunohistochemistry and Western blot. The LTLD+mEHT combination was more effective at inhibiting tumor growth than the free DOX and PLD, demonstrated by reductions in both the tumor volume and tumor weight. LTLD+mEHT resulted in the highest DOX accumulation in the tumor one hour after treatment. Tumor cell damage was associated with cC3 in the damaged area, and with a reduction in Ki67 in the living area. These changes were significantly the strongest in the LTLD+mEHT-treated tumors. The body weight loss was similar in all mice treated with any DOX formulation, suggesting no difference in toxicity. In conclusion, LTLD combined with mEHT represents a novel approach for DOX delivery into cancer tissue.