Murine Mammary Carcinoma Cells Research Articles

Abstract 4775: Unveiling caveolin-1's crucial role in breast cancer progression and metastasis: A molecular perspective

Abstract Background: Breast cancer, particularly its metastatic form, constitutes a significant portion of oncological mortality. Caveolin-1 (Cav-1), a caveolae structural protein, has been implicated in cellular processes such as proliferation, apoptosis, autophagy, and cellular motility (aberrant in breast cancer). With its dual role as a tumor suppressor and promoter, Cav-1's function in breast cancer pathophysiology requires elucidation for potential therapeutic targeting. Methods: We utilized a syngeneic mouse model to engineer Cav-1 deficient 4T1 murine mammary carcinoma cells which mirror the characteristics of stage IV breast cancer to investigate Cav-1's role in tumor growth and metastasis. We assessed cellular proliferation and lung metastasis, compared with wild-type controls, and performed molecular analyses to evaluate epithelial and mesenchymal marker expression, stemness-related markers, and autophagy-related gene expression. Results: Cav-1 ablation corresponded with a marked decrease in tumor proliferation and lung metastasis, indicating a suppressive role in tumor progression. Molecular characterization revealed downregulated epithelial markers (E-cadherin, β-catenin), transcription factors (TWIST1/2), and stem cell markers (NANOG, OCT4, SOX2, c-MYC, SALL4), with concurrent upregulation of the mesenchymal marker vimentin. Enhanced expression of autophagy-related genes (Beclin-1, ATG5, ATG16) was also observed, suggesting a disruption in autophagic pathways. Conclusion: Our findings underscore the critical role of Cav-1 in breast cancer progression, specifically its function in suppressing metastasis and modulating cellular phenotypes. Targeting Cav-1 could represent a novel therapeutic strategy to mitigate breast cancer growth and dissemination. Further research is warranted to explore the therapeutic potential of modulating Cav-1 in breast cancer treatment. Citation Format: Rizwana Begum, Shilpa Thota, Dhirendra Pratap Singh, Naveen Chintala, Abhishek Pandit, Biplov Sapkota, Joseph Francis. Unveiling caveolin-1's crucial role in breast cancer progression and metastasis: A molecular perspective [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 4775.

Abstract 3198: An analysis of breast cancer membrane lipid-modified nanoliposomes for enhanced targeting of triple-negative breast cancer

Abstract Background: Every breast cancer diagnosis is unique in its own way. Triple-negative breast cancer (TNBC), has a highly aggressive phenotype characterized by a rapid growth rate profile, increased risk of metastasis and recurrence. TNBC is devoid of epidermal growth factor receptor 2, estrogen, and progesterone receptors. All three are commonly present in other forms of breast cancer disease. The aim of this study was to develop a cell membrane lipid-extracted nanoliposomes (CLENs) for selective targeting compared to conventional nanoliposomal varieties. An additional objective was to investigate the role of microenvironment on targeting using cell model of TNBC, and representations of non-target tissue environments for comparison. Method: Lipid extracts (LE) obtained from 4T1, a murine mammary carcinoma cell line from a BALB/cfC3H mouse was used to formulate the CLENs (cell membrane lipid-extracted nanoliposomes) with different ratios of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a phospholipid used in most conventional drug delivery liposomes. Different lipid compositions and ratios were employed to evaluate cellular uptake in target, non-target and off-target cells. The target cell line, 4T1, off-target and non-target cell populations included CRL-2089, normal breast fibroblasts, SKBR3 a (human breast cancer), and A549 a (lung cancer) cell lines. Other studies involved determination of molecular charge and zeta potential of various nanoliposomal preparations and corresponding fluorescence studies in vitro. Results: The physicochemical properties of CLENs such as particle size and surface charge characteristics were vital when assessing benefit of the lipid-based preparations. The size of the CLENs typically ranged between 130 - 165 nm and zeta potential values were negatively-charged. The inclusion of 70 mol% of LE in preparation of 4T1-CLENs demonstrated increased cellular uptake and binding by the 4T1 target cells compared to the control DOPC (100%). The highest degree of selectivity was observed in early cell binding events, and with studies involving more extended incubation time points under different experimental conditions. Off-target cells demonstrated reduced uptake by comparison. Overall, 4T1-CLENs was most efficient when applied against intended target 4T1 cells, when compared to relevant control cell populations. In conclusion, studies performed to date suggest the notion that lipid extracts derived from TNBC improves targeting. Citation Format: Christiana Ayertey, Parmida Amid, Mohammed H. Almozain, Robert B. Campbell. An analysis of breast cancer membrane lipid-modified nanoliposomes for enhanced targeting of 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 3198.

A lipo-polymeric hybrid nanosystem with metal enhanced fluorescence for targeted imaging of metastatic breast cancer.

Cancer metastasis plays a major role in failure of therapeutic avenues against cancer. Owing to metastasis, nearly 70-80% of stage IV breast cancer patients lose their lives. Nanodrug delivery systems are playing a critical role in the therapy of metastatic cancer in the recent times. This paper reports the enhanced permeation and retention (EPR) based targeting of metastatic breast cancer using a novel nano lipo-polymeric system (PIR-Au NPs). The PIR-Au NPs demonstrated an increase in fluorescence by virtue of surface coating with gold, owing to the metal enhanced fluorescence phenomenon as reported in our earlier reports. Enhanced fluorescence of PIR-Au NPs was observed in murine mammary carcinoma cell line (4T1), as compared to free IR780 or IR780 loaded nanosystems (P-IR NPs), when incubated for same time at same concentrations, indicating its potential application for imaging and an enhanced bioavailability of IR780. Significant cell death was noted with photothermal mediated cytotoxicity in-vitro against breast cancer cells (MCF-7 and 4T1). An enhanced fluorescence was observed in the zebra fish embryos incubated with PIR-Au NPs. The enhanced permeation and retention (EPR) effect was seen with PIR-Au NPs in-vivo. A strong fluorescent signal was recorded in mice injected with PIR-Au NPs. The tumor tissue collected after 72 h, clearly showed a greater fluorescence as compared to other groups, indicating the plasmon enhanced fluorescence. We also demonstrated the EPR-based targeting of the PIR-Au NPs in-vivo by means of photothermal heat. This lipo-polymeric hybrid nanosystem could therefore be successfully applied for image-guided, passive-targeting to achieve maximum therapeutic benefits.

Cerium End-Deposited Gold Nanorods-Based Photoimmunotherapy for Boosting Tumor Immunogenicity.

Triple-negative breast cancer (TNBC) was closely related to high metastatic risk and mortality and has not yet found a targeted receptor for targeted therapy. Cancer immunotherapy, especially photoimmunotherapy, shows promising potential in TNBC treatment because of great spatiotemporal controllability and non-trauma. However, the therapeutic effectiveness was limited by insufficient tumor antigen generation and the immunosuppressive microenvironment. We report on the design of cerium oxide (CeO2) end-deposited gold nanorods (CEG) to achieve excellent near-infrared photoimmunotherapy. CEG was synthesized through hydrolyzing of ceria precursor (cerium acetate, Ce(AC)3) on the surface of Au nanorods (NRs) for cancer therapy. The therapeutic response was first verified in murine mammary carcinoma (4T1) cells and then monitored by analysis of the anti-tumor effect in xenograft mouse models. Under near-infrared (NIR) light irradiation, CEG can efficiently generate hot electrons and avoid hot-electron recombination to release heat and form reactive oxygen species (ROS), triggering immunogenic cell death (ICD) and activating part of the immune response. Simultaneously, combining with PD-1 antibody could further enhance cytotoxic T lymphocyte infiltration. Compared with CBG NRs, CEG NRs showed strong photothermal and photodynamic effects to destroy tumors and activate a part of the immune response. Combining with PD-1 antibody could reverse the immunosuppressive microenvironment and thoroughly activate the immune response. This platform demonstrates the superiority of combination therapy of photoimmunotherapy and PD-1 blockade in TNBC therapy.

Whey protein isolate attenuates depression-like behavior developed in a mouse model of breast tumor

Increasing evidence indicates that tryptophan (Trp) metabolism disturbance controls hippocampal 5-hydroxytryptamine (5-HT) and thereby affecting depression-like behavior, in which the gut microbiota (GM) might be involved. This study investigated the effect of Trp-rich whey protein isolate (WPI) on depressive-like behavior in 4T1 tumor-bearing mice. Female BALB/c mice were subcutaneously inoculated with murine 4T1 mammary carcinoma cells and received 2 g/kg of WPI by gavage daily for 21 days. The results showed that WPI exerted no significant effects on tumor weight and volume, but abrogated tumor-induced depression-like behavior, as evidenced by remarkably increased time and distance in the center of the open-field test, decreased immobility time in the tail suspension test, increased time and number of entries to the open arms in the elevated plus maze and sucrose preference. Moreover, WPI promoted the hippocampal Trp, 5-hydroxytryptophan (5-HTP), 5-HT, 5-hydroxyindoleacetic acid (5-HIAA) levels and inhibited kynurenine (Kyn) through up-regulating serotonin transporter (SERT) and down-regulating indoleamine 2, 3-dioxygenase (IDO). WPI showed an enriched microbial diversity indicated by increased Shannon index and decreased Simpson index, reduced the abundances of Proteobacteria, Rikenellaceae_RC9_gut_group, Alloprevotella and Prevotellaceae_UCG-001, and increased the abundance of unclassified_k__norank_d__Bacteria in tumor-bearing mice (P < 0.05). At level 3, WPI enhanced the function of microbial gene related to Trp metabolism in the KEGG pathways (P < 0.05). Our results suggest that WPI exhibits a potent antidepressant-like effect via the regulation of hippocampal Trp metabolism and alteration of GM composition and function, and it may be an effective prevention for cancer-related depression.

Isogenic Mammary Models of Intraductal Carcinoma Reveal Progression to Invasiveness in the Absence of a Non-Obligatory In Situ Stage.

In breast cancer, progression to invasive ductal carcinoma (IDC) involves interactions between immune, myoepithelial, and tumor cells. Development of IDC can proceed through ductal carcinoma in situ (DCIS), a non-obligate, non-invasive stage, or IDC can develop without evidence of DCIS and these cases associate with poorer prognosis. Tractable, immune-competent mouse models are needed to help delineate distinct mechanisms of local tumor cell invasion and prognostic implications. To address these gaps, we delivered murine mammary carcinoma cell lines directly into the main mammary lactiferous duct of immune-competent mice. Using two strains of immune-competent mice (BALB/c, C57BL/6), one immune-compromised (severe combined immunodeficiency; SCID) C57BL/6 strain, and six different murine mammary cancer cell lines (D2.OR, D2A1, 4T1, EMT6, EO771, Py230), we found early loss of ductal myoepithelial cell differentiation markers p63, α-smooth muscle actin, and calponin, and rapid formation of IDC in the absence of DCIS. Rapid IDC formation also occurred in the absence of adaptive immunity. Combined, these studies demonstrate that loss of myoepithelial barrier function does not require an intact immune system, and suggest that these isogenic murine models may prove a useful tool to study IDC in the absence of a non-obligatory DCIS stage-an under-investigated subset of poor prognostic human breast cancer.

The Blockade of Mitogen-Activated Protein Kinase 14 Activation by Marine Natural Product Crassolide Triggers ICD in Tumor Cells and Stimulates Anti-Tumor Immunity

Immunogenic cell death (ICD) refers to a type of cell death that stimulates immune responses. It is characterized by the surface exposure of damage-associated molecular patterns (DAMPs), which can facilitate the uptake of antigens by dendritic cells (DCs) and stimulate DC activation, resulting in T cell immunity. The activation of immune responses through ICD has been proposed as a promising approach for cancer immunotherapy. The marine natural product crassolide, a cembranolide isolated from the Formosan soft coral Lobophytum michaelae, has been shown to have cytotoxic effects on cancer cells. In this study, we investigated the effects of crassolide on the induction of ICD, the expression of immune checkpoint molecules and cell adhesion molecules, as well as tumor growth in a murine 4T1 mammary carcinoma model. Immunofluorescence staining for DAMP ectolocalization, Western blotting for protein expression and Z'-LYTE kinase assay for kinase activity were performed. The results showed that crassolide significantly increased ICD and slightly decreased the expression level of CD24 on the surface of murine mammary carcinoma cells. An orthotopic tumor engraftment of 4T1 carcinoma cells indicated that crassolide-treated tumor cell lysates stimulate anti-tumor immunity against tumor growth. Crassolide was also found to be a blocker of mitogen-activated protein kinase 14 activation. This study highlights the immunotherapeutic effects of crassolide on the activation of anticancer immune responses and suggests the potential clinical use of crassolide as a novel treatment for breast cancer.

Targeted delivery of epirubicin to breast cancer cells using poly-aptamer DNA nanocarriers prepared by the RCA method with multiple repeats of aptamers of FOXM1 and AS1411

Objective We evaluated the DNA nanocarriers synthesized by rolling circle amplification (RCA), composed of multiple repeats of AS1411 and FOXM1 aptamers for targeted epirubicin delivery to breast cancer cells. Methods Agarose gel electrophoresis and scanning electron microscopy were used to nanostructure characterizing. Drug loading and drug release were determined by fluorometry. Cytotoxicity comparison by MTT assay was applied among epirubicin, nanoparticle, and complex (nanoparticle carrying epirubicin) in L929 (normal murine fibroblast) and 4T1 (murine mammary carcinoma) cells. Cellular epirubicin internalization was assessed by flow cytometry and fluorescence imaging. In vivo studies in 4T1 tumor-bearing BALB/c mice were conducted by monitoring tumor volume, mouse weight, and mortality rate and measuring the accumulated epirubicin in organs. Results The negatively charged nanoparticles were under 200 nm and stable. Fifty microliters of 6 μM epirubicin was loaded in 50 μL nanoparticle. Epirubicin release at acidic pH was more. Complex compared with epirubicin, had more entry and cytotoxicity in target cells (p value ≤.01), higher therapeutic effect (p value ≤.001), and tumor drug accumulation. Conclusion The poly-aptamer nanocarriers have the characteristics of being safe, stable, efficient epirubicin loading, pH-dependent drug release, and tumor-targeting ability in vitro and in vivo.

IGF1R and HIF-1α Gene Silencing Inhibits Cancer Cell Growth

Background: According to several studies, hypoxia-inducible factor-1α (HIF-1α) and insulin-like growth factor 1 receptor (IGF1R) promote cancer progression and drug resistance. The overexpression of IGF1R and HIF-1 α is observed in various cancers, including breast and colon cancers. Thus, we tried to inhibit the progression of tumor cells by blocking IGF1R and HIF-1α. Methods: This study used Lipofectamine to transfect small interfering RNAs (siRNAs) into tumor cells. We treated murine mammary carcinoma (4T1) and murine colon carcinoma (CT26) cells with anti-IGF1R and anti-HIF-1 siRNAs. The real-time polymerase chain reaction (PCR) assay studied the impact of siRNA transfection on the mRNA levels of affected factors. Moreover, the viability of cancer cells was investigated by the MTT assay. Results: The investigations demonstrated that the mRNA levels of IGF1R and HIF-1α strongly reduced in tumor cell lines following siRNA transfection. Moreover, silencing IGF1R and HIF-1 additively downregulated cell viability in cancer cell lines. Conclusion: These findings imply that cancer combination therapy based on IGF1R and HIF-1α targeting may be a novel promising approach for the immunotherapy of breast and colorectal cancers; however, more study is required.

Peanut sprout tea extract inhibits lung metastasis of 4T1 murine mammary carcinoma cells by suppressing the crosstalk between cancer cells and macrophages in BALB/c mice.

As peanuts germinate, the content of the components beneficial to health, such as resveratrol, increases within the peanut sprout. This study examined whether the ethanol extract of peanut sprout tea (PSTE) inhibits breast cancer growth and metastasis. After orthotopically injecting 4T1 cells into BALB/c mice to induce breast cancer, 0, 30, or 60 mg/kg body weight/day of PSTE was administered orally. Angiogenesis-related protein expression in the tumors and the degree of metastasis were analyzed. 4T1 and RAW 264.7 cells were co-cultured, and reverse transcription polymerase chain reaction was performed to measure the crosstalk between breast cancer cells and macrophages. PSTE reduced tumor growth and lung metastasis. In particular, PSTE decreased matrix metalloproteinase-9, platelet endothelial cell adhesion molecule-1, vascular endothelial growth factor-A, F4/80, CD11c, macrophage mannose receptor, macrophage colony-stimulating factor, and monocyte chemoattractant protein 1 expression in the tumors. Moreover, PSTE prevented 4T1 cell migration, invasion, and macrophage activity in RAW 264.7 cells. PSTE inhibited the crosstalk between 4T1 cells and RAW 264.7 cells and promoted the macrophage M1 subtype while inhibiting the M2 subtype. These results suggest that PSTE blocks breast cancer growth and metastasis to the lungs. This may be because the PSTE treatment inhibits the crosstalk between mammary cancer cells and macrophages and inhibits the differentiation of macrophages into the M2 subtype.

Binary Pt/Te Nanoheterostructures with High Photothermal Conversion Efficiency and Anti-inflammatory Action for Enhanced Photothermal Therapy of 4T1 Breast Tumors Guided by Photoacoustic Imaging

Photothermal therapy is a powerful candidate for tumor treatment. However, photothermal therapy still faces some challenges, such as lacking photothermal agents with high photothermal conversion efficiency and undesirable inflammatory responses, which may result in tumor recurrence and therapeutic resistance. Here, the Pt/Te nanoheterostructures (PT) were synthesized by a simple hydrothermal reaction. The photothermal conversion efficiency was up to 51.84%. The outstanding photothermal conversion capacity of PT was attributed to the unique localized surface plasmon resonance frequency of metals and semiconductors and the increased circuit paths of electron transitions from nanoheterostructures. After coating with the murine mammary carcinoma (4T1) cell membrane, the camouflaged PT (mPT) exhibits excellent biocompatibility and effective homologous targeting capacity. Benefiting from antioxidative activity, mPT can efficiently scavenge inflammation-related reactive oxygen species and cytokines (such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) caused by hyperthermia to alleviate inflammation in vitro and in vivo. The in vitro and in vivo therapeutic results showed that mPT could effectively inhibit 4T1 breast tumors. In addition, the in vivo therapy could be guided by photoacoustic imaging. These results demonstrated that these multifunctional mPT provide a paradigm for biomimetic metal and semiconductor nanoheterostructures for enhanced photothermal therapy and anti-inflammatory action on tumors.

Interleukin-27 Disrupts the Crosstalk of Apoptotic Activities between 4T1 Breast Cancer Cells and M2 Macrophages

Introduction: Cytokine immunotherapy such as Interleukin-27 (IL-27) has been foreseen as a promising alternative anti-cancer treatment. Thus, this study aimed to investigate whether IL-27 gene therapy regulates crosstalk between breast cancer cells and macrophages in the sense of pro-apoptotic activities. Methods: This study has led to the development of recombinant pcDNA3.4-IL27. The recombinant pcDNA3.4-IL27 was transfected into 4T1 murine mammary carcinoma cells alone and co-culture of 4T1 with M2 macrophages. The successful expression of IL-27 in the cells were determine through the immunofluorescence staining and detection of CD206, M2 macrophages marker. Apoptotic effects of pcDNA3.4-IL27 were assessed through MTT assay, Annexin V flow cytometer analysis, and AO/PI dual staining. Results: Our findings shows that pcDNA3.4-IL27 has the ability to induce apoptosis in both of the cell group and performs better in the co-culture of 4T1 with M2 macrophages compared to 4T1 cells alone. PcDNA3.4-IL27 induced apoptosis through the altered cell morphology and reduction in the number of viable cells. Conclusion: These data demonstrate that pcDNA3.4-IL27 has the ability to induce apoptosis in both 4T1 cell alone and co-cultured 4T1 with M2 macrophages. Thus, could serve as a potential anti cancer candidate against breast cancer.

Enantiomeric NIR-II Emitting Rare-Earth-Doped Ag2 Se Nanoparticles with Differentiated In Vivo Imaging Efficiencies.

Here, chiral second near-infrared (NIR-II) emitting rare-earth doped silver selenide nanoparticles (R- or S-Ag2 Se:Nd/Yd/Er NPs) were fabricated, exhibiting circular dichroism peak at 850 nm and fluorescence peak at 1550 nm, with 145.7-fold enhanced intensity compared to the reported Ag2 Se NPs. Compared with S-Ag2 Se:Nd/Yd/Er NPs, imaging efficiency of R-Ag2 Se:Nd/Yd/Er NPs in living cells was significantly improved due to a higher cellular uptake rate and 927.7-fold higher affinity. Furthermore, R-Ag2 Se:Nd/Yd/Er NPs reached at the tumor 2-fold faster than S type of NPs in vivo. We discover that chirality leads to differences in the affinity between chiral Ag2 Se:Nd/Yd/Er NPs and cluster of differentiation 44 (CD44) onto the surface of murine mammary carcinoma cell to cause different in vivo imaging efficiency. These results reveal that chiral Ag2 Se:Nd/Yd/Er NPs have high photoluminescence intensity and high in vivo imaging efficiency reflecting wide applications in biomedical diagnosis.

Enantiomeric NIR‐II Emitting Rare‐Earth‐Doped Ag2Se Nanoparticles with Differentiated In Vivo Imaging Efficiencies

AbstractHere, chiral second near‐infrared (NIR‐II) emitting rare‐earth doped silver selenide nanoparticles (R‐ or S‐Ag2Se:Nd/Yd/Er NPs) were fabricated, exhibiting circular dichroism peak at 850 nm and fluorescence peak at 1550 nm, with 145.7‐fold enhanced intensity compared to the reported Ag2Se NPs. Compared with S‐Ag2Se:Nd/Yd/Er NPs, imaging efficiency of R‐Ag2Se:Nd/Yd/Er NPs in living cells was significantly improved due to a higher cellular uptake rate and 927.7‐fold higher affinity. Furthermore, R‐Ag2Se:Nd/Yd/Er NPs reached at the tumor 2‐fold faster than S type of NPs in vivo. We discover that chirality leads to differences in the affinity between chiral Ag2Se:Nd/Yd/Er NPs and cluster of differentiation 44 (CD44) onto the surface of murine mammary carcinoma cell to cause different in vivo imaging efficiency. These results reveal that chiral Ag2Se:Nd/Yd/Er NPs have high photoluminescence intensity and high in vivo imaging efficiency reflecting wide applications in biomedical diagnosis.

Tumor-derived extracellular vesicles for the active targeting and effective treatment of colorectal tumors in vivo

Colorectal cancer remains one of the main causes of cancer-related deaths worldwide. Although numerous nanomedicine formulations have been developed to tackle the disease, their low selectivity still limits effective therapeutic outcomes. In this study, we isolated extracellular vesicles (EVs) from CT26 colorectal cancer cells and 4T1 murine mammary carcinoma cells, loaded them with the chemotherapeutic agent (doxorubicin, DOX). Then we evaluated the cellular uptake of the extracellular vesicles both in 2D monolayer and 3D tumor spheroid setups using confocal laser scanning microscope and flow cytometry. In vivo tumor homing of the extracellular vesicles was verified on CT26 tumor bearing BALB/c mice using in vivo imaging system. Finally, in vivo therapeutic effects were evaluated and compared using the same animal models treated with five doses of EV formulations. CT26-EV-DOX exhibited excellent biocompatibility, a high drug-loading capacity, controlled drug release behavior, and a high capability for targeting colorectal cancer cells. In particular, we verified that CT26-EV-DOX could preferentially be up taken by their parent cells and could effectively target and penetrate 3D tumor spheroids resembling colorectal tumors in vivo in comparison with their 4T1 derived EV partner. Additionally, treatment of colorectal tumor-bearing BALB/c mice with of CT26-EV-DOX significantly inhibited the growth of the tumors during the treatment course. The developed CT26-EV-DOX nanoparticles may present a novel and effective strategy for the treatment of colorectal cancer.

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.

Poly(I:C) transfection induces a pro-inflammatory cascade in murine mammary carcinoma and fibrosarcoma cells

ABSTRACT Germline-encoded pattern recognition receptors [PRRs] in mammalian cells function in the detection of molecular patterns associated with pathogen invasion or cellular damage. A PRR subset is activated by the atypical presence and location of double-stranded RNA [dsRNA] or its synthetic analogue polyinosinic-polycytidylic acid [poly(I:C)], triggering pro-inflammatory signalling and death in many cell types. Poly(I:C) has been tested as a sole or combination cancer therapy in preclinical studies and clinical trials. The purpose of this study was to evaluate the effects of poly(I:C) transfection via electroporation on cell lines from a cancer of epithelial origin, 4T1 mammary carcinoma, and a cancer of mesenchymal origin, WEHI 164 fibrosarcoma. The effects of the poly(I:C) delivery on cell metabolism implicate the induction of cell death. A pro-inflammatory response was demonstrated by mRNA upregulation and the secretion of Type I interferon and several cytokines and chemokines. The mRNAs of dsRNA sensor DExD/H-box helicase 58/retinoic acid-inducible gene I protein [Ddx58/RIG-I] and sensor/co-sensor DEAH-box helicase 9 [Dhx9] were not regulated, but the mRNAs of RNA sensors toll-like receptor 3 [TLR3], interferon-induced with helicase C domain 1/melanoma differentiation-associated protein 5 [Ifih1/MDA5] and Z-DNA binding protein 1 [Zbp1] and co-sensors DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 [Ddx60] and interferon-inducible protein 204 [Ifi204] were upregulated in both cell lines. The mRNAs encoding signalling pathways components were present or upregulated in both cell types. These data demonstrate that RNA sensing effects can be amplified by electroporation delivery, potentially expanding the practicality of this immunotherapeutic approach.

Abstract 1773: Overcoming paclitaxel resistance in triple-negative breast cancer using a novel barcoding technology

Abstract Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype for which chemotherapy remains a part of standard treatment. Although pathologic complete response (pCR) after neoadjuvant chemotherapy, including paclitaxel (PTX), is associated with good outcomes, 50-60% of TNBC patients do not experience pCR and suffer poor long-term outcomes, often due to chemotherapy resistance. Identifying and analyzing the tumor cells responsible for chemotherapy resistance will lead to improved treatment strategies. We developed a model of TNBC resistance to neoadjuvant PTX using Met1 murine mammary carcinoma cells. To study clonal dynamics in response to PTX we developed a barcoding method, SunCatcher. First, 31 single-cell derived clonal populations were generated from parental Met1 cells. Each clone was infected with a lentiviral vector containing a unique DNA barcode detectable by qPCR. All barcoded clones (BCs) were mixed in equivalent numbers to generate a BC pool that we confirmed captures the parental Met1 heterogeneity and tumor growth kinetics. First, PTX responses were tested in vitro. The BC pool had an IC50 of ~100 nM, while individual BCs ranged in IC50 from 5 nM to 25 µM. We maintained the BC pool in 100 nM PTX for 39 days (termed long-term PTX; LTP), at which point it became clonal for BC25, suggesting that BC25 (IC50 of 100 nM) is uniquely PTX resistant, despite being the most proliferative and Zeb1hi/EpCAMlow clone in vitro. BC25 was not detected in the control-treated BC pool at 39 days. Next, the BC pool was orthotopically injected into FVB/NJ mice and treated the mice with 20 mg/kg PTX on days 7, 11, and 15. At the day 18 experimental endpoint, tumor volume significantly decreased by 56% in response to PTX compared to control. BC25 composition increased from 7.7% (controls) to 15% (PTX treated) within the tumors at endpoint. We also injected BC25 and LTP cells alone and administered the same PTX regimen once tumors reached 50 mm3. Both BC25 and LTP tumors were unresponsive to PTX and had a longer latency period (35d) than BC pool (10d). We performed a drug screen of 2313 compounds spanning FDA-approved cancer therapeutics on the LTP cells to identify compounds to target the PTX-resistant clone. HDAC inhibitors were the most potent class of hits and one, Panobinostat, killed LTP cells with an IC50 of 4.3 nM. Utilizing SunCatcher, we identified a unique PTX-resistant TNBC subclone that represents residual disease associated with poor long-term outcome. Typical in vivo experiments would have reached ethical endpoint before BC25 had a chance to grow, given its long latency, therefore this PTX-resistant clone would not have been identified. This is an important finding because therapeutic resistance can emerge after a protracted period, longer than typical pre-clinical experiments. Further work will explore mechanisms of resistance and the potential for combination therapies to prevent recurrent disease. Citation Format: Milos Spasic, Qiuchen Guo, Adam Maynard, Gregory Goreczny, Adrienne Waks, Sara Tolaney, Elizabeth Mittendorf, Sandra McAllister. Overcoming paclitaxel resistance in triple-negative breast cancer using a novel barcoding technology [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 1773.

Functional, Aromatic, and Fluorinated Monothiosemicarbazones: Investigations into Their Structures and Activity toward the Gallium-68 Incorporation by Microwave Irradiation.

We report on the synthesis and spectroscopic characterization of a new series of coordinating monothiosemicarbazones incorporating aromatic backbones, featuring O/N/S donor centers monosubstituted with different aliphatic, aromatic, fluorinated, and amine-functionalized groups at their N centers. Their ability to bind metal ions such as Zn(II) and Ga(III) was explored, and the formation of two different coordination isomers of the Zn(II) complex was demonstrated by X-ray diffraction studies using synchrotron radiation. These studies showed the planar geometry for the coordinated mono(thiosemicarbazone) ligand and that the metal center can adopt either a heavily distorted tetrahedral Zn center (placed in an N/S/S/N environment, with CN = 4) or a pseudo-octahedral geometry, where the Zn(II) center is in the O/N/S/S/N/O environment, and CN = 6. Furthermore, 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT) assays and cellular imaging in living cells were subsequently performed in two different cancer cell lines: PC-3 (a standard cell line derived from a bone metastasis of a stage IV prostate cancer) and EMT6 (a commercial murine mammary carcinoma cell line). The radiolabeling of new functional and aromatic monothiosemicarbazones with either gallium-68 (under pH control) or fluorine-18 is discussed. The potential of this class of compounds to act as synthetic scaffolds for molecular imaging agents of relevance to positron emission tomography was evaluated in vitro, and the cellular uptake of a simultaneously fluorinated and [68Ga]-labeled mono(thiosemicarbazone) was investigated and is reported here.

Synthesis and Characterization of Size- and Charge-Tunable Silver Nanoparticles for Selective Anticancer and Antibacterial Treatment

Advances in the research of nanoparticles (NPs) with controlled charge and size are driven by their potential application in the development of novel technologies and innovative therapeutics. This work reports the synthesis, characterization, and comprehensive biological evaluation of AgNPs functionalized by N,N,N-trimethyl-(11-mercaptoundecyl) ammonium chloride (TMA) and trisodium citrate (TSC). The prepared AgNPs were well characterized in terms of their morphological, spectroscopic and functional properties and biological activities. The implementation of several complementary techniques allowed not only the estimation of the average particle size (from 3 to 40 nm depending on the synthesis procedure used) but also the confirmation of the crystalline nature of the NPs and their round shape. To prove the usefulness of these materials in biological systems, cellular uptake and cytotoxicity in microbial and mammalian cells were determined. Positively charged 10 nm Ag@TMA2 revealed antimicrobial activity against Gram-negative bacteria with a minimum inhibitory concentration (MIC) value of 0.17 μg/mL and complete eradication of Escherichia coli (7 logs) for Ag@TMA2 at a concentration of 0.50 μg/mL, whereas negatively charged 10 nm Ag@TSC1 was effective against Gram-positive bacteria (MIC = 0.05 μg/mL), leading to inactivation of Staphylococcus aureus at relatively low concentrations. In addition, the largest 40 nm Ag@TSC2 was shown to exhibit pronounced anticancer activity against murine colon carcinoma (CT26) and murine mammary gland carcinoma (4T1) cells cultured as 2D and 3D tumor models and reduced toxicity against human HaCaT keratinocytes. Among the possible mechanisms of AgNPs are their ability to generate reactive oxygen species, which was further evaluated in vitro and correlates well with cellular accumulation and overall activity of AgNPs. Furthermore, we confirmed the anticancer efficacy of the most potent Ag@TSC2 in hiPSC-derived colonic organoids and demonstrated that the NPs are biocompatible and applicable in vivo. A pilot study in BALB/c mice evidenced that the treatment with Ag@TSC2 resulted in temporary (>60 days) remission of CT26 tumors.