Murine Breast Cancer Model Research Articles

Biogenic synthesis of gold nanoparticles using dual extract of tulsi-Vinca for breast cancer tumor regression in mice.

Aim: This work aims to synthesize the gold nanoparticles (GNPs) using a dual extract of tulsi and Vinca (T+V-Gold) for breast cancer tumor regression. Methods: The GNPs were synthesized and characterized for their microscopic, spectroscopicand crystalline properties. Further, the GNPs were investigated for invitro and invivo studies for the treatment of the 4T1-induced triple-negative breast cancer murine model. Results: The GNPs for 4T1 tumor-challenged mice resulted in delayed tumor development and lower tumor burden, with T+V-Gold demonstrating the highest prevention of tumor spread. The antitumor effect of T+V-Gold is highly significant in the glutathione family antioxidants glutathione S-transferase and glutathione in tumor tissue samples. Conclusion: The bioefficacy and anticancer outcomes of T+V-Gold nanoformulation can be used as therapeutic agents and drug-delivery vehicles.

Repeated electroporation enhances the responsiveness to cisplatin-based intra-tumoral chemotherapy in murine breast cancer: revival of the electro-chemotherapy concept

Background and aims: Electroporation demonstrated certain modulable actions on tumoral cell membrane permeability to increase the intracellular bioavailability of chemotherapeutic drugs. The current in vivo study aimed to investigate the synergic effect of concomitant electroporation application to the intratumoral administration of cisplatin on murine invasive ductal adenocarcinoma breast cancer. Methods: The fragments of the extracted tumor were implanted subcutaneously in healthy female Balb/C mice. Having reached the determined tumoral volume, the mice were randomly divided into five groups, including control, intratumoral cisplatin injection, tumoral electroporation application, electrochemotherapy (ECT), and double course ECT. The normalized tumoral volume and the inhibition ratios were calculated during a 30-day follow-up period. The data were tested by ANONA, and a statistically significant level was set at P<0.05. Results: The inhibition ratio was significantly different between the intra-tumoral cisplatin administration and tumoral electroporation application groups compared to the control group (P<0.05). ECT displayed superior results in comparison to the two later groups (P<0.05). The double-course ECT group even represented a significant difference compared to the ECT group (P<0.05). Conclusion: Concomitant ECT to the cisplatin intratumoral administration indicated contributive anti-tumoral impacts in an in vivo murine model of invasive ductal adenocarcinoma breast cancer. ECT promises further applications to overcome the occurrence of therapeutic resistance to chemotherapeutic drugs.

THU482 The Cholesterol Metabolite, 27-hydroxycholesterol, Enhances The Secretion Of Cancer Promoting Extracellular Vesicles By Impairing Lysosomal Integrity

Abstract Disclosure: A. Das Gupta: None. N. Krawczynska: None. H. Vidana Gamage: None. H. Kim: None. J. Park: None. J.E. Sorrells: None. S.A. Boppart: None. E.R. Nelson: None. Breast cancer is a highly prevalent cancer among women and mortality is typically associated with metastatic disease. Elevated circulating cholesterol has been associated with breast cancer recurrence in pre-clinical as well as clinical models. The cholesterol metabolite, 27-hydroxycholesterol (27HC), has been identified to establish an immunosuppressive tumor microenvironment via Liver X Receptor (LXR) in myeloid immune cells. Interestingly, 27HC treatment of different myeloid immune cell types results in increased secretion of extracellular vesicles (EVs) with altered cargo. Importantly, EVs from 27HC treated myeloid cells promote tumor growth and metastasis in murine models of breast cancer. Given that these altered EVs are involved in cancer progression, it is imperative that we understand how their biogenesis and secretion are regulated. The formation of EVs involves maturation of endosomes to form multivesicular bodies containing intraluminal vesicles. These MVBs are either targeted to the lysosomes for degradation or directed towards the plasma membrane to release EVs. Immunofluorescence microscopy experiments revealed that 27HC increases the size and number of CD63+ multivesicular bodies (MVBs) but does not alter EEA1+ endosomes in RAW 264.7 cells. Quantitative PCR analysis revealed no significant changes in mRNA expression of genes associated with EV biogenesis machinery. However, we observed that 27HC increased the size and number of LAMP1+ lysosomes. This led to the hypothesis that 27HC increases EV secretion by skewing MVBs away from lysosomal degradation and towards secretion as EVs by i) impairing lysosomal integrity, and/or ii) altering the post-translational modifications on the MVBs. To study the effect of 27HC on lysosomal function, we found that 27HC increased in lysosomal pH, suggesting a loss of lysosomal integrity. Treatment of cells with a lysosomal proton pump inhibitor phenocopies the 27HC mediated increase in EV secretion. Post-translational modifications such as ISGylation and NEDDylation have been previously implicated in modulating EV secretion and cargo, and we have observed that 27HC decreases overall ISGylation and NEDDylation of proteins in cells. Thus, 27HC increases the size of multivesicular bodies, impairs lysosomal integrity, and alters levels of ISG- and NEDDylated proteins in cells, and a combination of these modulations leads to the increase in EV secretion. Collectively, our studies reveal novel mechanisms by which EV secretion is modulated by 27HC. By extension, this axis represents a novel avenue for intervening in EV secretion and ultimately in the development of breast cancer therapeutics. Funding: Department of Defense Breast Cancer Research Program Era of Hope Scholar Award (BC200206), and National Cancer Institute (R01CA234025). Presentation: Thursday, June 15, 2023

Imaging Tumor-Targeting Bacteria Using 18F-Fluorodeoxysorbitol Positron Emission Tomography.

Microbial-based cancer treatments are an emerging field, with multiple bacterial species evaluated in animal models and some advancing to clinical trials. Noninvasive bacteria-specific imaging approaches can potentially support the development and clinical translation of bacteria-based cancer treatments by assessing the tumor and off-target bacterial colonization. 18F-Fluorodeoxysorbitol (18F-FDS) positron emission tomography (PET), a bacteria-specific imaging approach, was used to visualize an attenuated strain of Yersinia enterocolitica, currently in clinical trials as a microbial-based cancer treatment, in murine models of breast cancer. Y. enterocolitica demonstrated excellent 18F-FDS uptake in in vitro assays. Whole-body 18F-FDS PET demonstrated a significantly higher PET signal in tumors with Y. enterocolitica colonization compared to those not colonized, in murine models utilizing direct intratumor or intravenous administration of bacteria, which were confirmed using ex vivo gamma counting. Conversely, 18F-fluorodeoxyglucose (18F-FDG) PET signal was not different in Y. enterocolitica colonized versus uncolonized tumors. Given that PET is widely used for the management of cancer patients, 18F-FDS PET could be utilized as a complementary approach supporting the development and clinical translation of Y. enterocolitica-based tumor-targeting bacterial therapeutics.

Emodin reduces surgical wounding-accelerated tumor growth and metastasis via macrophage suppression in a murine triple-negative breast cancer model.

It has been suspected that tumor resection surgery itself may accelerate breast cancer (BC) lung metastasis in some patients. Emodin, a natural anthraquinone found in the roots and rhizomes of various plants, exhibits anticancer activity. We examined the perioperative use of emodin in our established surgery wounding murine BC model. Emodin reduced primary BC tumor growth and metastasis in the lungs in both sham and surgical wounded mice, consistent with a reduction in proliferation and enhanced apoptosis (primary tumor and lungs). Further, emodin reduced systemic inflammation, most notably the number of monocytes in the peripheral blood and reduced pro-tumoral M2 macrophages in the primary tumor and the lungs. Consistently, we show that emodin reduces gene expression of select macrophage markers and associated cytokines in the primary tumor and lungs of wounded mice. Overall, we demonstrate that emodin is beneficial in mitigating surgical wounding accelerated lung metastasis in a model of triple-negative BC, which appears to be mediated, at least in part, by its actions on macrophages. These data support the development of emodin as a safe, low-cost, and effective agent to be used perioperatively to alleviate the surgery triggered inflammatory response and consequential metastasis of BC to the lungs.

The activation of SIRT1 by resveratrol reduces breast cancer metastasis to lung through inhibiting neutrophil extracellular traps

Neutrophil extracellular traps (NETs) play a crucial role in breast cancer metastasis. However, the therapeutic target of NETs in breast cancer metastasis is still unknown. Using a natural metabolite library and single-cell sequencing data analysis, we identified resveratrol (RES), a polyphenolic natural phytoalexin, and agonist of silent information regulator-1 (SIRT1) that suppressed NETs formation after cathepsin C (CTSC) treatment. In vivo, RES significantly hindered breast cancer metastasis in a murine orthotopic 4T1 breast cancer model. Serum levels of myeloperoxidase-DNA and neutrophil elastase-DNA in mouse breast cancer model were significantly lower after RES treatment. Correspondingly, the tumour infiltrated CD8+T cells in the lungs increased after the treatment. Mechanistically, RES targets SIRT1 in neutrophils and significantly inhibits the citrullination of histones H3, which is essential for chromatin decondensation and NETs formation. Furthermore, we identified that the NETs were suppressed by RES in bone marrow neutrophils after CTSC treatment, while specific deficiency of SIRT1 in neutrophils promoted NETs formation and breast cancer to lung metastasis. Thus, our results revealed that RES could be potentially identified as a viable therapeutic drug to prevent neutrophil cell death and breast cancer metastasis.

Reshaping Macrophage Polarization Potential Enhances Antitumor Immune Response to Radiation Therapy

Although radiation therapy (RT) remains a cornerstone in the treatment of breast cancer, many trials combining RT with immune checkpoint blockade (ICB) have failed to demonstrate benefit in solid tumors including breast cancer. Maximal efficacy of RT relies on the generation of antitumor immunity following treatment which largely consists of cytotoxic T cells and macrophages. Broad depletion of macrophages modestly enhances tumor responses to RT suggesting that they can shape RT-induced antitumor immunity. Although IL4 signaling through GATA-3 is known to polarize T cells into the protumor Th2 phenotype, such central drivers of macrophage polarization are not well established. Given that macrophages abundantly express IL4 receptor, we hypothesized that GATA-3 may direct the transition of macrophages to M2/alternative phase and that genetic ablation of GATA-3 in macrophages can enhance antitumor immunity by arresting macrophage transition to an M2-like pro-tumor state. We generated a macrophage specific GATA-3 KO mouse model (mG3KO) driven by the LysM-Cre promoter. Using a syngeneic orthotopic murine model of breast cancer (EO771), we evaluated the differential effect of RT (16Gy x 1) in WT and mG3KO mice. Multiparametric flow cytometry was performed to investigate the immune changes within the tumor microenvironment on day 3, day 5 and day 10 after RT. T cell depletion was performed using antibodies to CD4 and CD8 by intraperitoneal injections to understand the role of adaptive immunity in the response to RT in WT and mG3KO mice. We found that mG3KO mice bearing advanced EO771 tumors demonstrated significantly improved tumor regression compared to WT mice (p<0.001), which translated to increased overall survival. In vitro characterization of bone-marrow derived macrophages from mG3KO and WT mice suggest that macrophages with ablated GATA-3 expressed increased levels of iNOS and decreased levels of Arginase (Arg-1), consistent with an M1-like phenotype. Immune profiling of the tumors also revealed that mGATA-3 KO animals have significant enrichment of CD8+ T cells in the tumor milieu post RT and these CD8+ T cells express higher amounts of interferon gamma (p<0.001) and Granzyme B (p<0.0015) than their WT counterparts. Using neutralizing antibodies to deplete CD8+ T cells, we show that anti-tumor effects in the mG3KO mice were abolished, suggesting that mG3KO macrophages impact survival, at least, in part by enhancing cytotoxic CD8+T cells. Studies are currently ongoing to reveal the detailed mechanism of GATA-3 ablation in improving the efficacy of RT. Our data indicates that GATA-3 is a central regulator of macrophage polarization in response to RT. Further, directed ablation of GATA-3 appears to drive macrophages towards an M1-like phenotype, which enhances T cell recruitment to irradiated tumors. These data suggest that the antitumor efficacy of RT can be prolonged by targeting GATA-3-dependent signaling within myeloid cells.

Non-Redundant Mechanisms of Immune Resistance to Radiotherapy Converge on Innate Immunity

Despite evidence of preclinical synergy between radiotherapy (RT) and immune checkpoint blockade (ICB), randomized trials of RT/ICB have demonstrated limited benefit in solid tumors. We performed single-cell RNA sequencing (scRNA-seq) and CITE-seq (cellular indexing of transcriptomes and epitopes) to address the discordance between preclinical and clinical data. We hypothesized that multiple orthogonal inhibitory immune pathways restrain the local and systemic efficacy of RT beyond T-cell oriented immune checkpoints. We used the EO771 syngeneic murine model of breast cancer to characterize the immune tumor microenvironment following RT with or without ICB. RT (16 Gy x 1) was delivered using the X-RAD SmART platform with CT image guidance. Neutralizing antibodies (anti-PD-1/Ly6G/Gr-1/CD47) were delivered by intraperitoneal injections. scRNA-seq analysis were performed by Seurat and BBrowser (BioTuring). We found that adaptive ICB (anti-PD-1) reprogrammed the immune response to RT by promoting an M1-like interferon-primed state (ISG15, CXCL10) in tumor associated macrophages (TAMs) and by increasing the late recruitment of intratumoral neutrophils. Given that neutrophils may drive resistance to RT in other models, we evaluated the effect of intratumoral neutrophil depletion using anti-Ly6G or anti-Gr-1 on the antitumor efficacy of RT/ICB. Both neutrophil depletion strategies led to enhanced tumor control and improved survival in advanced EO771 tumors compared to RT/ICB alone (P<0.001). In parallel to this approach, we found that TAMs upregulated several innate immune checkpoints including SIRPα in response to RT. Disruption of the SIRPα-CD47 interaction by anti-CD47 antibodies similarly enhanced the antitumor efficacy of RT/ICB by improving tumor control and survival (P<0.001). Using scRNA-seq and unbiased clustering, we found that anti-CD47 eliminated an entire cluster of chronically inflamed TAMs, characterized by pro-inflammatory markers (IL1A, NOS2) and chemokines (CCL3, CXCL1/2/3). Anti-CD47 also reduced intratumoral neutrophils by eliminating a cluster of pathologically activated neutrophils, termed myeloid-derived suppressor cells (PMN-MDSCs) that expressed several markers of ferroptosis (TFRC, PTGS2, SLC3A2). Consistent with the potent immunosuppressive capacity of PMN-MDSCs, we found that anti-CD47 increased tumor-infiltrating lymphocytes including central memory TCF7+ T cells and CD19+ B cells. Lastly, by inference and analysis of cell-cell communication (CellChat), we found that anti-CD47 strengthened the interactions between TAMs and CD8+ T cells compared to RT/ICB alone. Our data collectively indicate that resistance to RT/ICB in the EO771 model Is driven by innate immune cells including neutrophils and chronically inflamed TAMs. Targeted disruption of the CD47-SIRPα axis is a promising approach to overcoming immune resistance by reprogramming TAMs and eliminating PMN-MDSCs.

Tumor Cell-Intrinsic PD-L1 Effects on Radiation-Induced Locoregional Antitumor Immunity

Targeting PD-L1 is a beneficial strategy to reinvigorate antitumor immunity, however variable response and resistance are challenging and suggest the need for multimodality approaches. Tumor cell-intrinsic PD-L1 signals also regulate non-canonical pathogenic pathways that may impact treatment resistance. Ionizing radiation (IR) can induce antitumor immunity and has demonstrated therapeutic synergy with immunotherapy in some cases, however tumor-driven immunologic mechanisms affecting clinical outcomes remain incompletely understood. In this study, we investigated the impact of tumor cell-intrinsic PD-L1 signals on IR-induced locoregional immune response and tumor control. We used orthotopic B16-F10 melanoma (WT-B16) and 4T1 triple negative breast cancer (WT-4T1) murine tumor models, as well as PD-L1 disabled variants (KO) generated by CRISPR/Cas9, implanted bilaterally. IR (10 Gy) was targeted at one tumor alone to evaluate both direct and indirect IR effects based on tumor PD-L1 status. We evaluated response by tumor volume (TV) measurements, flow cytometry of tumor-infiltrating lymphocytes (TILs) and tumor draining lymph nodes (TDLNs) in both irradiated and unirradiated compartments, and granzyme B (GZB) PET imaging to assess functional in vivo changes. Chemokine-based multiplex assays were used to assess cell lines receiving IR (4Gy) and ex vivo tumor lysates and serum. IR-induced local tumor control was not significantly affected based on tumor PD-L1 status, however deactivation of tumor cell PD-L1 enhanced IR-induced regional tumor control. Unirradiated WT tumors in mice harboring irradiated KO but not irradiated WT tumors demonstrated a significant mean reduction in TV with instances of complete distant tumor regression. PET imaging demonstrated a nearly 2-fold higher concentration of GZB in KO versus WT tumors, in line with known locally immunosuppressive effects of tumor PD-L1. Remarkably, GZB levels were >1.5-fold higher in unirradiated WT tumors in mice harboring an irradiated KO versus WT tumor, which correlated with a 50% increase in PD-1+CD8+ T cells. Higher levels of CD62+CD44- naïve CD4+ (4-fold) and CD8+ (2-fold) memory T cells were seen in TDLNs of irradiated KO versus WT tumors. Cytokine levels positively correlated with immune recruitment and activation status, as CXCL10, CCL2 and CCL5 were significantly upregulated in PD-L1 KO versus WT tumors cells. Results from this study demonstrate cell-intrinsic PD-L1 inhibits IR-induced locoregional immune activation and frequency of regional tumor control, with clinical implications including therapeutic targeting of tumor cell-intrinsic PD-L1 signals to enhance IR-induced immunogenicity, utility of IR based on tumor PD-L1 status particularly in the metastatic setting, and immunotherapy combinations. Future studies investigating mechanisms of resistance to IR-induced immune activation to enhance responsiveness are warranted.

Leptin/lipopolysaccharide-treated dendritic cell vaccine improved cellular immune responses in an animal model of breast cancer

Purpose In dendritic cells (DCs), leptin as an immune-regulating hormone, increases the IL-12 generation whereas it reduces the IL-10 production, thus contributing to TH1 cell differentiation. Using a murine model of breast cancer (BC), we evaluated the impacts of the Leptin and/or lipopolysaccharide (LPS)-treated DC vaccine on various T-cell-related immunological markers. Materials and methods Tumors were established in mice by subcutaneously injecting 7 × 105 4T1 cells into the right flank. Mice received the DC vaccines pretreated with Leptin, LPS, and both Leptin/LPS, on days 12 and 19 following tumor induction. The animals were sacrificed on day 26 and after that the frequency of the splenic cytotoxic T lymphocytes (CTLs) and TH1 cells; interferon gamma (IFN-γ), interleukin 12 (IL-12) and tumor growth factor beta (TGF-β) generation by tumor lysate-stimulated spleen cells, and the mRNA expression of T-bet, FOXP3 and Granzyme B in the tumors were measured with flow cytometry, ELISA and real-time PCR methods, respectively. Results Leptin/LPS-treated mDC group was more efficient in blunting tumor growth (p = .0002), increasing survival rate (p = .001), and preventing metastasis in comparison with the untreated tumor-bearing mice (UT-control). In comparison to the UT-control group, treatment with Leptin/LPS-treated mDC also significantly increased the splenic frequencies of CTLs (p < .001) and TH1 cells (p < .01); promoted the production of IFN-γ (p < .0001) and IL-12 (p < .001) by splenocytes; enhanced the T-bet (p < .05) and Granzyme B (p < .001) expression, whereas decreased the TGF-β and FOXP3 expression (p < .05). Conclusion Compared to the Leptin-treated mDC and LPS-treated mDC vaccines, the Leptin/LPS-treated mDC vaccine was more effective in inhibiting BC development and boosting immune responses against tumor.

In Situ Insonation of Alkaline Buffer Containing Liposomes Leads to a Net Improvement of the Therapeutic Outcome in a Triple Negative Breast Cancer Murine Model.

Breast cancer is characterized by an acidic micro-environment. Acidic extracellular pH gives cancer cells an evolutionary advantage, hence, neutralization of the extracellular pH has been considered as a potential therapeutic strategy. To address the issue of systemic pH alteration, an approach based on the targeted delivery of the buffering solution to the tumor region is investigated. The method relies on the use of low frequency ultrasound and sono-sensitive liposomes loaded with buffers at alkaline pH (LipHUS). After the i.v. injection of LipHUS, the application of ultrasound (US) at the sites of the pathology induces a local increase of pH that results highly effective in i) inhibiting primary tumor growth, ii) reducing tumor recurrence after surgery, and iii) suppressing metastases' formation. The experiments are carried out on a triple negative breast cancer mouse model. The results obtained demonstrate that localized and triggered release of bicarbonate or PBS buffer from sonosensitive liposomes represents an efficient therapeutic tool for treating triple-negative breast cancer. This approach holds promise for potential clinical translation.

GK-1 effectively reduces angiogenesis and prevents T cell exhaustion in a breast cancer murine experimental model

Breast cancer is the leading malignancy in women worldwide, both in terms of incidence and mortality. Triple-negative breast cancer (TNBC) is the type with the worst clinical outcomes and with fewer therapeutic options than other types of breast cancer. GK-1 is a peptide that in the experimental model of the metastatic 4T1 breast cancer has demonstrated anti-tumor and anti-metastatic properties. Herein, GK-1 (5 mg/kg, i.v.) weekly administrated not only decreases tumor growth and the number of lung macro-metastases but also lung and lymph nodes micro-metastases. Histological analysis reveals that GK-1 reduced 57% of the intra-tumor vascular areas, diminished the leukemoid reaction's progression, and the spleens' weight and length. A significant reduction in VEGF-C, SDF-1, angiopoietin-2, and endothelin-1 angiogenic factors was induced. Moreover, GK-1 prevents T cell exhaustion in the tumor-infiltrating lymphocytes (TILs) decreasing PD-1 expression. It also increased IFN-γ and granzyme-B expression and the cytotoxic activity of CD8+ TILs cells against tumor cells. All these features were found to be associated with a better antitumor response and prognosis. Altogether, these results reinforce the potential of GK-1 to improve the clinical outcome of triple-negative breast cancer immunotherapy. Translation research is ongoing towards its evaluation in humans.

Facile preparation of silver based radiosensitizers via biomineralization method for enhanced in vivo breast cancer radiotherapy

To solve the traditional radiotherapy obstacles, and also to enhance the radiation therapy efficacy various radiosensitizers have been developed. Radiosensitizers are promising agents that under X-ray irradiation enhance injury to tumor tissue by accelerating DNA damage. In this report, silver-silver sulfide nanoparticles (Ag-Ag2S NPs) were synthesized via a facile, one-pot and environmentally friendly biomineralization method. Ag-Ag2S was coated with bovine serum albumin (BSA) in situ and applied as an X-ray sensitizer to enhance the efficiency of radiotherapy. Also, folic acid (FA) was conjugated to Ag-Ag2S@BSA to impart active targeting capability to the final formulation (Ag-Ag2S@BSA-FA). Prepared NPs were characterized by transmission electron microscopes (TEM), scanning electron microscope (SEM), dynamic light scattering (DLS), ultraviolet–visible spectroscopy (UV–Vis), X-ray diffraction analysis (XRD), and X-ray photoelectron spectroscopy (XPS) techniques. Results show that most of the NPs have well-defined uniform Janus structures. The biocompatibility of the NPs was then evaluated both in vitro and in vivo. A series of in vitro assays were performed on 4T1 cancer cells to evaluate the therapeutic efficacy of the designed NPs. In addition, the radio-enhancing ability of the NPs was tested on the 4T1 breast cancer murine model. MTT, live and dead cell staining, apoptosis, ROS generation, and clonogenic in vitro assays demonstrated the efficacy of NPs as radiosensitizers in radiotherapy. In vivo results as well as H&E staining tumor tissues confirmed tumor destruction in the group that received Ag-Ag2S@BSA-FA NPs and exposed to X-ray. The results showed that prepared tumor-targeted Ag-Ag2S@BSA-FA NPs could be potential candidates as radiosensitizers for enhanced radiotherapy.

Exploration and Biological Evaluation of 1,3-Diamino-7H-pyrrol[3,2-f]quinazoline Derivatives as Dihydrofolate Reductase Inhibitors.

Dihydrofolate reductase (DHFR), a core enzyme of folate metabolism, plays a crucial role in the biosynthesis of purines and thymidylate for cell proliferation and growth in both prokaryotic and eukaryotic cells. However, the development of new DHFR inhibitors is challenging due to the limited number of scaffolds available for drug development. Hence, we designed and synthesized a new class of DHFR inhibitors with a 1,3-diamino-7H-pyrrol[3,2-f]quinazoline derivative (PQD) structure bearing condensed rings. Compound 6r exhibited therapeutic effects on mouse models of systemic infection and thigh infection caused by methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300. Moreover, methyl-modified PQD compound 8a showed a strong efficacy in a murine model of breast cancer, which was better than the effects of taxol. The findings showcased in this study highlight the promising capabilities of novel DHFR inhibitors in addressing bacterial infections as well as breast cancer.

Calorie restriction outperforms bariatric surgery in a murine model of obesity and triple-negative breast cancer.

Obesity promotes triple-negative breast cancer (TNBC), and effective interventions are urgently needed to break the obesity-TNBC link. Epidemiologic studies indicate that bariatric surgery reduces TNBC risk, while evidence is limited or conflicted for weight loss via low-fat diet (LFD) or calorie restriction (CR). Using a murine model of obesity-driven TNBC, we compared the antitumor effects of vertical sleeve gastrectomy (VSG) with LFD, chronic CR, and intermittent CR. Each intervention generated weight and fat loss and suppressed tumor growth relative to obese mice (greatest suppression with CR). VSG and CR regimens exerted both similar and unique effects, as assessed using multiomics approaches, in reversing obesity-associated transcript, epigenetics, secretome, and microbiota changes and restoring antitumor immunity. Thus, in a murine model of TNBC, bariatric surgery and CR each reverse obesity-driven tumor growth via shared and distinct antitumor mechanisms, and CR is superior to VSG in reversing obesity's procancer effects.

Intratumoral heterogeneity after targeted therapy in murine cancer models with differing degrees of malignancy

IntroductionConventional morphologic and volumetric assessment of treatment response is not suitable for adequately assessing responses to targeted cancer therapy. The aim of this study was to evaluate changes in tumor composition after targeted therapy in murine models of breast cancer with differing degrees of malignancy via non-invasive magnetic resonance imaging (MRI). Materials and methodsMice bearing highly malignant 4T1 tumors or low malignant 67NR tumors were treated with either a combination of two immune checkpoint inhibitors (ICI, anti-PD1 and anti-CTLA-4) or the multi-tyrosine kinase inhibitor sorafenib, following experiments with macrophage-depleting clodronate-loaded liposomes and vessel-stabilizing angiopoietin-1. Mice were imaged on a 9.4 T small animal MRI system with a multiparametric (mp) protocol, comprising T1 and T2 mapping and diffusion-weighted imaging. Tumors were analyzed ex vivo with histology. Results and discussionsAll treatments led to an increase in non-viable areas, but therapy-induced intratumoral changes differed between the two tumor models and the different targeted treatments. While ICI treatment led to intratumoral hemorrhage, sorafenib treatment mainly induced intratumoral necrosis. Treated 4T1 tumors showed increasing and extensive areas of necrosis, in comparison to 67NR tumors with only small, but also increasing, necrotic areas. After either of the applied treatments, intratumoral heterogeneity, was increased in both tumor models, and confirmed ex vivo by histology. Apparent diffusion coefficient with subsequent histogram analysis proved to be the most sensitive MRI sequence. In conclusion, mp MRI enables to assess dedicated therapy-related intratumoral changes and may serve as a biomarker for treatment response assessment.

Oncolytic myxoma virus is effective in murine models of triple negative breast cancer despite poor rates of infection

Oncolytic viruses are being heavily investigated as novel methods to treat cancers; however, predicting their therapeutic efficacy remains challenging. The most commonly used predictive tests involve determining the invitro susceptibility of a tumor's malignant cells to infection with an oncolytic agent. Whether these tests are truly predictive of invivo efficacy, however, remains unclear. Here we demonstrate that a recombinant, oncolytic myxoma virus shows efficacy in two murine models of triple negative breast cancer despite extremely low permissivity of these models to viral infection. These data demonstrate that invitro infectivity studies are not an accurate surrogate for therapeutic efficacy and suggest that other tests need to be developed.

Regulation of IGF1R by MicroRNA-15b Contributes to the Anticancer Effects of Calorie Restriction in a Murine C3-TAg Model of Triple-Negative Breast Cancer.

Calorie restriction (CR) inhibits triple-negative breast cancer (TNBC) progression in several preclinical models in association with decreased insulin-like growth factor 1 (IGF1) signaling. To investigate the impact of CR on microRNAs (miRs) that target the IGF1/IGF1R pathway, we used the spontaneous murine model of TNBC, C3(1)/SV40 T-antigen (C3-TAg). In C3-TAg mice, CR reduced body weight, IGF1 levels, and TNBC progression. We evaluated the tumoral expression of 10 miRs. CR increased the expression of miR-199a-3p, miR-199a-5p, miR-486, and miR-15b. However, only miR-15b expression correlated with tumorigenicity in the M28, M6, and M6C C3-TAg cell lines of TNBC progression. Overexpressing miR-15b reduced the proliferation of mouse (M6) and human (MDA-MB-231) cell lines. Serum restriction alone or in combination with low levels of recombinant IGF1 significantly upregulated miR-15b expression and reduced Igf1r in M6 cells. These effects were reversed by the pharmacological inhibition of IGFR with BMS754807. In silico analysis using miR web tools predicted that miR-15b targets genes associated with IGF1/mTOR pathways and the cell cycle. Our findings suggest that CR in association with reduced IGF1 levels could upregulate miR-15b to downregulate Igf1r and contribute to the anticancer effects of CR. Thus, miR-15b may be a therapeutic target for mimicking the beneficial effects of CR against TNBC.

Liposome-encapsulated zoledronate increases inflammatory macrophage population in TNBC tumours

BackgroundTumour associated macrophages (TAMs) are important players in breast tumour progression and metastasis. Clinical and preclinical evidence suggests a role for zoledronate (ZOL) in breast cancer metastasis prevention. Further, zoledronate is able to induce inflammatory activation of monocytes and macrophages, which can be favourable in cancer treatments. The inherent bone tropism of zoledronate limits its availability in soft tissues and tumours. In this study we utilised an orthotopic murine breast cancer model to evaluate the possibility to use liposomes (EMP-LIP) to target zoledronate to tumours to modify TAM activation. MethodsTriple-negative breast cancer 4T1 cells were inoculated in the 4th mammary fat pad of female Balb/c mice. Animals were divided according to the treatment: vehicle, ZOL, EMP-LIP and liposome encapsulated zoledronate (ZOL-LIP). Treatment was done intravenously (with tumour resection) and intraperitoneally (without tumour resection). Tumour growth was followed by bioluminescence in vivo imaging (IVIS) and calliper measurements. Tumour-infiltrating macrophages were assessed by immunohistochemical and immunofluorescence staining. Protein and RNA expression levels of inflammatory transcription factors and cytokines were measured by Western Blotting and Taqman RT-qPCR. ResultsLiposome encapsulated zoledronate (ZOL-LIP) treatment suppressed migration of 4T1 cell in vitro. Tumour growth and expression of the angiogenic marker CD34 were reduced upon both ZOL and ZOL-LIP treatment in vivo. Long-term ZOL-LIP treatment resulted in shift towards M1-type macrophage polarization, increased CD4 T cell infiltration and activation of NF-κB indicating changes in intratumoural inflammation, whereas ZOL treatment showed similar but non-significant trends. Moreover, ZOL-LIP had a lower bisphosphonate accumulation in bone compared to free ZOL. ConclusionResults show that the decreased bisphosphonate accumulation in bone promotes the systemic anti-tumour effect of ZOL-LIP by increasing inflammatory response in TNBC tumours via M1-type macrophage activation.

Multiparametric chemical exchange saturation transfer MRI detects metabolic changes in breast cancer following immunotherapy

BackgroundWith metabolic alterations of the tumor microenvironment (TME) contributing to cancer progression, metastatic spread and response to targeted therapies, non-invasive and repetitive imaging of tumor metabolism is of major importance. The purpose of this study was to investigate whether multiparametric chemical exchange saturation transfer magnetic resonance imaging (CEST-MRI) allows to detect differences in the metabolic profiles of the TME in murine breast cancer models with divergent degrees of malignancy and to assess their response to immunotherapy.MethodsTumor characteristics of highly malignant 4T1 and low malignant 67NR murine breast cancer models were investigated, and their changes during tumor progression and immune checkpoint inhibitor (ICI) treatment were evaluated. For simultaneous analysis of different metabolites, multiparametric CEST-MRI with calculation of asymmetric magnetization transfer ratio (MTRasym) at 1.2 to 2.0 ppm for glucose-weighted, 2.0 ppm for creatine-weighted and 3.2 to 3.6 ppm for amide proton transfer- (APT-) weighted CEST contrast was conducted. Ex vivo validation of MRI results was achieved by 1H nuclear magnetic resonance spectroscopy, matrix-assisted laser desorption/ionization mass spectrometry imaging with laser postionization and immunohistochemistry.ResultsDuring tumor progression, the two tumor models showed divergent trends for all examined CEST contrasts: While glucose- and APT-weighted CEST contrast decreased and creatine-weighted CEST contrast increased over time in the 4T1 model, 67NR tumors exhibited increased glucose- and APT-weighted CEST contrast during disease progression, accompanied by decreased creatine-weighted CEST contrast. Already three days after treatment initiation, CEST contrasts captured response to ICI therapy in both tumor models.ConclusionMultiparametric CEST-MRI enables non-invasive assessment of metabolic signatures of the TME, allowing both for estimation of the degree of tumor malignancy and for assessment of early response to immune checkpoint inhibition.