Mouse Breast Cancer Cell Line Research Articles

Abstract PO5-24-01: Narazaciclib’s differential targets and kinase inhibitory activity contribute to the enhanced inhibition of tumor growth in preclinical models

Abstract Despite improvement of outcomes for HR+, HER2- metastatic breast cancer by cyclin dependent kinase 4 and 6 (CDK4/6) inhibitors, safety concerns and disease progression raise a critical need to identify novel approaches. Narazaciclib (ON123300), a novel CDK4/6i, designed to enhance efficacy and safety by its multi-targeted kinase inhibitor activity. Narazaciclib is in Ph I trials; NCT04739293 and CXHL1900340; studying different administration regimens and in endometrial cancer in combination with letrozole (NCT05705505). Our study explored the activity of narazaciclib and its metabolite ON1232580 in comparisons to the FDA approved CDK4/6i and identify additional targets engaged by narazaciclib. To identify direct and secondary targets engaged by narazaciclib and palbociclib, proteome wide Cellular Thermal Shift Assay (CETSA) and Integrative Inferred Kinase Activity (INKA) analysis were performed. Both CETSA and INKA analysis identified additional targets engaged by narazaciclib compared to palbociclib in both MDA-MB-231 lysates and intact cells such as BUB1, CHEK1, AURKA, GSK3α and GSK3β. In TNBC patients with BUB1 overexpression, bioinformatics analysis indicates a low survival correlation. Molecular docking data showed a higher affinity of narazaciclib with BUB1 compared to palbociclib and abemaciclib. The differential activity of narazaciclib was explored by using cohorts of human and mouse breast cancer cell lines and cell based assays. Comparison of narazaciclib’s in vitro IC50 profile to other CDK4/6i was studied against a panel of 370 kinases (HotSpot) and Kd values were determined by KINOMEscan. Intracellular IC50 kinase values were determined by NanoBret technology. Narazaciclib and abemaciclib were found to be the most promiscuous in vitro kinase inhibitors and ribociclib the most specific. Abemaciclib and narazaciclib had similar profiles against the CDK family members. Kd values of CDK4/cyclinD1 binding show similar trends; abemaciclib (0.08 nM), narazaciclib (0.18 nM), palbociclib (0.75 nM) and ribociclib (1.3 nM). Although narazaciclib displayed nM IC50 values in the in vitro assays against many CDKs, narazaciclib was very specific in cellular kinase assays with highest activity against CDK4/6, CSF1R and NUAK1. A stronger inhibition of cell proliferation, as well as induction of apoptosis and senescence were detected in narazaciclib and its metabolite ON1232580 treated PYMT cells, a murine mammary carcinoma model which recapitulates luminal BC subtype, compared to the other CDK4/6i. Both narazaciclib and ON1232580 enhanced CCL5 and CXCL10 mRNA levels, while a higher reduction of PD-L1 and pRb protein levels and a promoting effect on the H2D1 and B2M mRNA levels was shown in narazaciclib and its metabolite treated PYMT cells. Narazaciclib showed significant synergy with anti-PD1 in the EMT-6 syngeneic breast cancer model. A differential effect of narazaciclib on the expression levels of 62 cytokines was detected compared to abemaciclib and palbociclib. Lastly, inhibition of autophagy sensitizes cancer cells to both ON123300 and ON1232580, and induces irreversible cell proliferation inhibition, providing a novel therapeutic approach. Our findings identify important differences generated from assay models studying kinase inhibition, binding and pathway engagement. Understanding the role of the differential targets engaged by narazaciclib, the potential enhanced antitumor immunity and the sensitization by autophagy inhibitors to cell death, will guide future clinical studies. Citation Format: Petros Kechagioglou, Debomita Chakraborty, Camille Dupont, Hajime Yurugi, Ute Distler, Stefan Tenzer, Alexey Chernobrovkin, Kristina Riegel, Julianne Mooz, Mahil Lamber, Volker Dötsch, Stephen Cosenza, Steven Fruchtman, Krishnaraj Rajalingam. Narazaciclib’s differential targets and kinase inhibitory activity contribute to the enhanced inhibition of tumor growth in preclinical models [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-01.

Abstract PO2-24-10: Anti-Tumorigenic Immuno-Microenvironment by AB-329, a Potent and Selective AXL inhibitor, combined with paclitaxel in Triple-Negative Breast Cancer Preclinical Model

Abstract BACKGROUND The Gas6/AXL signaling pathway plays a substantial role in cancer cell survival, proliferation, immunosuppression, and metastasis. Elevated AXL expression is associated with advanced cancer stages and decreased overall survival rates, underscoring its potential as a therapeutic target for metastatic cancers. We investigated this possibility by utilizing AB-329, a selective small molecule AXL kinase inhibitor, to treat triple-negative breast cancer (TNBC). TNBC, typified by the lack of ER, PR, and HER2 expression, frequently displays mesenchymal characteristics and carries a significant risk of metastasis and an immunosuppressive tumor microenvironment. METHODS We assessed AXL protein expression in 28 human and 11 mouse breast cancer cell lines using Western blotting. The Microarray database was utilized to examine AXL gene expression in 40 human breast cancer cells. The Sulforhodamine B proliferation and soft agar assays measured the antiproliferative effect of AB-329 in TNBC cell lines. The in vitro anti-metastatic impact of AB-329 was evaluated through migration and invasion assays. We appraised the anti-tumor effects of AB-329 in combination with paclitaxel using 4T1 and E0771-LMB mouse TNBC xenograft models. We used FACS and CIBERSORTx to perform immune cell type profiling in E0771-LMB xenograft tumor tissues. To understand AB-329’s immunomodulatory effects in tumors, single-cell RNA sequencing was conducted in a humanized mouse model with SUM149 human TNBC xenografts. RESULTS Microarray data revealed higher AXL expression in TNBC cells compared to non-TNBC cells. Western blotting supported this, showing higher AXL protein expression, particularly in basal and mesenchymal subtypes of TNBC cells, compared to luminal breast cancer cells. AB-329 presented moderate antiproliferative effects as a monotherapy in the TNBC cell lines tested, with half-maximal inhibitory concentrations exceeding 5 µM. Nevertheless, combining AB-329 with paclitaxel resulted in significant growth inhibition in all tested TNBC cell lines (p < 0.05). Importantly, AB-329 alone could inhibit TNBC migration and invasion at a concentration of 1 µM (p < 0.01) and showed enhanced inhibition rates when paired with paclitaxel (p < 0.05). The combination of AB-329 and paclitaxel considerably reduced tumor growth in the 4T1 and E0771-LMB xenograft models compared to paclitaxel or AB-329. Total RNA sequencing and CIBERSORTx analysis indicated that the AB-329 and paclitaxel combination increased Natural Killer (NK) cells’ infiltration into E0771-LMB mouse TNBC xenograft tumors compared to single agent-treated tumors. A nearly 2.5-fold increase in NKT cell infiltration was also detected with AB-329 treatment compared to Control in SUM149 human TNBC xenografts. CONCLUSIONS Our preclinical data suggest that AB-329, an AXL inhibitor, could synergize effectively with paclitaxel in TNBC treatment, notably inhibiting tumor cell proliferation and migration while enhancing NK cell infiltration. Intriguingly, we observed an increased infiltration of NKT cells into tumors following AB-329 treatment, which may promote an anti-tumorigenic immuno-microenvironment. These results underscore the necessity for further investigation into the role of AB-329 in modulating the tumor immuno-microenvironment through AXL pathway inhibition and accentuate the potential for combining AB-329 with immunotherapy. Citation Format: Dileep Reddy Rampa, Jon Fuson, Huey Liu, Max Pan, Naoto Ueno, Jangsoon Lee. Anti-Tumorigenic Immuno-Microenvironment by AB-329, a Potent and Selective AXL inhibitor, combined with paclitaxel in Triple-Negative Breast Cancer Preclinical Model [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-10.

Effects of Metformin on JNK Signaling Pathway and PD-L1 Expression in Triple Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Metformin has been shown to have the potential to inhibit the proliferation of malignant cells. This study aimed to investigate the regulatory effect of metformin on the expression of programmed death protein ligand 1(PD-L1) and mechanisms in TNBC. Mouse breast cancer cell line 4T1 was co-cultured with metformin, and the effect of metformin on cell proliferation was detected by MTT assay. The effect of metformin on the expression of JNK, RSK2 and CREB was detected by MAPK pathway protein chip. BALB/c mice were inoculated with 4T1 cells with knockdown/overexpression of C-Jun N-terminal kinase (JNK), and administered with metformin. The weight of tumor tissue was observed at the end of the experiment. The expression of PD-L1 in tumor cells was observed by immunofluorescence staining and the level of INF-γwas quantitatively determined by ELISA. Metformin inhibited the viability of 4T1 cells and increased the phosphorylation of JNK to reduce the phosphorylation of RSK2 and CREB. Metformin and JNK knockdown reduced the expression of PD-L1 in tumor cells, but there was no significant difference in the weight of tumor tissue. Metformin can reduce the level of INF-γ in tumor tissues, but JNK has no effect. Metformin can inhibit the expression of PD-L1 in triple-negative breast cancer mice and improve the tumor microenvironment, but does not reduce the size of the tumor.

Abstract 4051: Optimizing breast cancer therapy by modulating oxygen consumption

Abstract Immune checkpoint blockade (ICB) has achieved great clinical responses in multiple cancers. However, only a subset of patients with triple negative breast cancer (TNBC), a highly hypoxic tumor, respond to ICB. In TNBC, the immune checkpoints PD-1/PD-L1 and adenosine receptor (A2aR) are often activated to escape anti-tumor responses. These two immune checkpoints are sensitive to hypoxia. We propose that blocking tumor oxygen consumption using deferiprone (DFP), phenformin (Phen) and metformin (Met) will improve the efficacy of PD-1/PD-L1 and A2aR blockade. We generated two mouse breast cancer cell lines (4T1 and E0771) expressing green fluorescent protein (GFP) fused to Firefly luciferase under the control of the hypoxia reporter element (HRE). This construct, which we called HRE/TGL, serves as a hypoxia reporter, with GFP expression and luciferase activity increasing in hypoxic conditions. To induce hypoxia in vitro, we used two approaches: 1) we incubated cells in a hypoxia chamber (3% oxygen), and 2) treated cells with cobalt chloride, which chemically mimics hypoxia by inducing the expression of the hypoxia master regulator HIF1α. Both 4T1-HRE/TGL and E0771-HRE/TGL cells exhibited increased GFP expression and luciferase activity within hypoxia conditions. We orthotopically implanted these cells into mice. We measured luciferase activity in vivo using the IVIS spectrum imaging system overtime after tumor implantation. We observed high luciferase activity in 4T1-HRE/TGL tumors. Next, we examined the effects of DFP, Phen and Met on cell hypoxic responses by performing immunoblotting against HIF1α and phosphorylated AMPK (p-AMPK) on 4T1 and E0771 cells. We observed that, unlike Phen or Met, DFP increased HIF1α and p-AMPK expression. Binding of HIF1 proteins to HREs is known to activate the expression of the HRE promoter. To confirm our results, we treated 4T1-HRE/TGL and E0771-HRE/TGL cells with low (10μM) or high (100μM) doses of the drugs in vitro. We observed that, while DFP increased GFP expression and luciferase activity, Phen had no consistent measurable effect. We observed similar results in vivo. Finally, we examined the effect of DFP, Phen and Met either alone or in combination with anti-PD-1 on tumor progression and survival in mice bearing 4T1 or E0771 tumors. Contrary to what expected, we did not observe better tumor control and survival after treatment with these drugs. In addition, DFP, Phen and Met failed to improve the anti-tumor effect of anti-PD-1 treatment in both TNBC models. Future experiments will focus on charactering the adenosine pathway in vivo in 4T1 and E0771 tumors and examine how drugs targeting A2aR and oxygen consumption (DFP, Phen and Met) influence the activation of immune cells within the tumor microenvironment and potentially combinate these two strategies to improve their anti-tumor efficacy. Citation Format: Anais Assouvie, Sadna Budhu, Mayuresh Mane, Mamadou Bah, Juan Zurita, Inna Serganova, Soe Min, Jason Koutcher, Vladimir Ponomarev, Jedd Wolchok, Taha Merghoub. Optimizing breast cancer therapy by modulating oxygen consumption [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 4051.

Abstract 2630: Identifying most promising combination therapies for immune suppressive breast cancer using a combined in vitro/in vivo approach

Abstract Combining epigenetic therapy with immunotherapy is a promising strategy to address the complexities of cancer biology. However, it's important to note that the field is still evolving, and more research is needed to understand the optimal combinations, timing, and patient populations that will benefit the most from these approaches. We evaluated a combination of two different checkpoint inhibitor therapies (anti-CTLA4 and anti-PD1) with two different hypomethylating agents (5-azacytidine, AZA and Decitabine, DEC) in vitro and in vivo using a mouse breast cancer cell line 4T1 known to be resistant towards checkpoint inhibition (CPi). Beside tumor cell killing and tumor volume measurement, cytokine expression and immune cell phenotyping was determined. In a co-culture assay with autologous T cells and 4T1 cells, antitumor efficacy weas increased in all combination arms using AZA but not in combination with DEC. The combination of AZA with anti-CTLA4 was significantly better than the respective monotherapies (oneway ANOVA, p< 0.001). This effect was observed as well in the combination with anti-PD1 and AZA, however it was less pronounced (oneway ANOVA, p< 0.01). Of note, AZA restored the CTLA4 expression on CD8+ T cells in vitro, which was reduced by co-culture with tumor cells alone. In the follow-up in vivo experiment we focus on the combination of both CPi with AZA on different treatment schedules in the combination arms. The combination of anti-CTLA4 and AZA in a staggered treatment schedule displayed significant efficacy compared to control (oneway ANOVA, p< 0.05). The implantation of 4T1 reduced the number of T cells in bone marrow (BM), peripheral blood (PB) and spleen of the mice, confirming the immune-suppressive nature of the breast cancer line. This effect was restored under treatment with AZA in monotherapy and combination with CPi. Interestingly AZA reduced the number of immunosuppressive MDSC in BM, spleen and in tumor tissue. Vice versa NK cells were increased in BM, spleen and tumor tissue under treatment with AZA. AZA reduced the number of circulation tumor cells in PB and increased as already shown in vitro the percentage of CD8/CTLA4++ in PB. The PCA analysis of 36 mouse cytokines in vivo displayed two main clusters separated mainly by the presence of absence of AZA in the treatment arms. The combination arms which displayed the highest efficacy showed an increase in CXCL1, G-CSF, IL-6, LIF, M-CSF, MCP-1 (CCL2) and MCP-3 (CCL7) and a decrease of IFN-gamma, CXCL2. This is in line with the more inflammatory pattern of the tumor infiltrating lymphocytes in those groups. In summary, we have strong evidence that AZA influences the immune landscape of breast cancer towards a more inflammatory subtype. The combination with CPi was superior to the monotherapy. In addition, our data indicate that the treatment schedule of the combination might have a crucial influence on efficacy. Citation Format: Hannah Silberzahn, Mattias Bleisch, Anne Löhr, Volker Knauff, Kanstantsin Lashuk, Julia B. Schueler. Identifying most promising combination therapies for immune suppressive breast cancer using a combined in vitro/in vivo approach [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 2630.

A cancer cell nanotherapy method based on GHz film bulk acoustic resonator and nanoscale CaO2.

Sonodynamic therapy (SDT) is an emerging cancer treatment method in recent years. However, the ultrasound signal utilized for SDT is usually located at a low-frequency spectrum (<2MHz), and in the field of SDT research, few studies have focused on the exploration and development of ultrasound frequency. Studies have shown that the GHz-level ultrasound can increase cell membrane permeability and have a negligible effect on cell vitality. Herein, we reported the study of a GHz thin film bulk acoustic resonator as an ultrasound source for synergistic treatment with nanoscale calcium peroxide (CaO2). It was discovered that this ultrasound source ultimately achieved an efficient therapeutic outcome on mouse breast cancer cell line 4T1. Such GHz-level ultrasound application in SDT is of high significance to broaden the cognition and application scope of SDT.

Abstract B019: Deciphering the role of homologous recombination deficiency mutations in the response to combined immune checkpoint and PARP inhibitors

Abstract Background: Over 2 million women develop breast cancer globally every year despite immense advances in disease biology, diagnostic strategies and novel treatment options. The deeper knowledge of disease biology and genomic landscape has fostered a broader mechanistic approach to patients and their therapeutic choices. Many preclinical and clinical studies suggest a benefit of combining PARP inhibitors (PARPi) and immune checkpoint inhibitors (ICPi) in patients with homologous recombination deficiency (HRD) intact and mutated cancers. However, similar to the varied benefits of either drug class based on tissue and mutational context, the benefits of such combination are strongly variable based on tumor type, tissue and mutation context. Methods: To study how select HRD mutations respond to a combination of a PARPi inhibitor with an ICPi, we used CRISPR engineering to functionally impair or repress BRCA1, BRCA2, ATM, CHEK2, PALB2 in different mouse breast cancer cell lines. EMT6 and 4T1 cells were engineered to expressed deactivated cas9 nuclease (dcas9) fused to the repressor domain Kruppel-associated- box (KRAB) and mcherry florescent protein. The dcas9-KRAB-mcherry breast cancer cells were further engineered to express BFP-tagged guide RNAs targeting selected genes under a doxycycline inducible tet-on system. After establishing functional loss/repression of the targeted HRD mutations, cells were then implanted in the mammary fat pad of balb/cJ mice and treated with PARPi and ICPi to determine the ICPi, PARPi and their combination induced changes in inflammatory signals and their association with therapy response. Results: Induced suppression of BRCA1 and PALB2 in these breast cancer cells showed exquisite sensitivity to PARPi (IC50 0.01 uM and 0.098 uM) and diminishing sensitivity to PARPi in BRCA2 and ATM (IC50 0.16 uM and IC50 0.28 uM). In contrast, inducible suppression of CHEK2 showed resistance to PARPi versus parental EMT6 cells (IC50 13.9 uM versus 5.3 uM). Evaluation of changes in expression levels of PDL1 and select chemokines including CCL2, CCL5, CXCL9, CXCL10 and CXCL11 suggest a differential response to PARPi depending on specific HRD mutations. HRD mutations and tissue-context will be further characterized in combination therapy of ICPi and PARPi. Conclusion: Using inducible suppression of HRD function in an immune competent mouse breast cancer model helps to understand the differential role of individual HRD mutations (BRCA1, BRCA2, ATM, CHEK2, PALB2) in altering inflammatory signals in response to PARPi and ICI. This data may guide the clinical development of such combination in breast cancer and other cancers. Citation Format: Sibapriya Chaudhuri, Scott Thomas, Luika Timmerman, Pamela Munster. Deciphering the role of homologous recombination deficiency mutations in the response to combined immune checkpoint and PARP inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B019.

A hyaluronic-based prodrug with aggregation-induced emission for drug delivery and cellular imaging

A novel drug molecule (R6) is conjugated to hyaluronic acid (HA) to form a pH-responsive prodrug with aggregation-induced emission (AIE) property. Owing to its amphiphilic nature, the prodrug could self-assemble into nanoparticles (NP) in an aqueous solution. This formulation thereby gave rise to AIE of the R6 moieties which resided in the NP core. The polymer could release the drug at the tumor microenvironment (TME) acidic condition (99% release after 72 h), while remaining stable at the physiological pH. In addition, fluorescence signals by AIE from the NP could be used for cellular imaging. The hyaluronic acid shell can target the overexpressed CD44 receptors in cancer cells, which gives the NP active targeting property. The prodrug showed toxicity against the mouse breast cancer cell line 4T1 while being harmless to the L929 fibroblast cells. Fluorescence microscopy images confirmed the imaging ability of the NP in 4T1 cells. The HA-R6 polymer prodrug promises to be a versatile pH-sensitive drug delivery platform.

A Novel Bromophenol Compound from Leathesia nana Inhibits Breast Cancer in a Direct Tumor Killing and Immunotherapy Manner.

Considering the resistance and toxicity of traditional chemotherapeutic drugs, seeking potential candidate for treating breast cancer effectively is a clinical problem that should be solved urgently. Natural products have attracted extensive attention, owing to their multi-target advantages and low toxicity. In the current study, the effects of XK-81, a novel bromophenol compound extracted from Leathesia nana, on breast cancer, and its underlying mechanisms, were explored. Firstly, data from in vitro experiments indicated that 4T-1, one of common mouse breast cancer cell lines, was a XK-81-susceptible cell line, and ferroptosis was the major death manner in response to XK-81 treatment, which was evidenced by increasing intracellular Fe2+ and ROS level with condensed mitochondrial membrane densities, as well as decreasing the protein expressions of SLC7A11 and GPX4. In vivo, XK-81 suppressed the growth of 4T-1 breast-tumor in both BALB/C mice and zebrafish. Obviously, XK-81 decreased the protein expression of SLC7A11 and GPX4 in tumor tissues, hinting at the occurrence of ferroptosis. Moreover, XK-81 increased CD8+ T cells and NK cells numbers and regulated M1/M2 macrophage ratio in tumor tissues, indicating XK-81's immunotherapeutic effect. Additionally, the secretions of immune-related cytokines, including TNF-α, IL-1β, and IL-12, were elevated with XK-81 stimulation in RAW 264.7 cells. Intriguingly, compared with doxorubicin-induced heart damage, XK-81 demonstrated the therapeutic advantage of little cardiotoxicity on the heart. XK-81 demonstrated potential antitumor advantage by both directly inducing ferroptosis-mediated death of tumor cells and immunization.

Reconstituted Lipid Nanoparticles from Cells/Tissues for Drug Delivery in Cancer.

Nanomedicine represents a promising way to devise better drug delivery systems (DDSs), and the development of cell/tissue-based lipid carriers is a promising strategy. In this study, the author proposes the concept of reconstituted lipid nanoparticles (rLNPs) and offers a facile preparation method. The results demonstrated that the preparation of ultrasmall (∼20 nm) rLNPs can be highly reproducible from both cells (a mouse breast cancer cell line, 4T1) and tissue (mouse liver tissue). As a selected model platform, rLNPs derived from mouse liver tissue can be further labeled with imaging molecules (indocyanine green and coumarin 6) and modified with targeting moiety (biotin). Moreover, rLNPs were proved to be highly biocompatible and able to load various drugs, such as doxorubicin hydrochloride (Dox) and curcumin (Cur). Most importantly, Dox-loaded rLNPs (rLNPs/Dox) exerted good in vitro and in vivo anticancer performances. Therefore, rLNPs might be a potential versatile carrier for the construction of different DDSs and treatment of a variety of diseases.

Novel Tetrahydro-[1,2,4]triazolo[3,4-a]isoquinoline Chalcones Suppress Breast Carcinoma through Cell Cycle Arrests and Apoptosis.

Chalcones are interesting anticancer drug candidates which have attracted much interest due to their unique structure and their extensive biological activity. Various functional modifications in chalcones have been reported, along with their pharmacological properties. In the current study, novel chalcone derivatives with the chemical base of tetrahydro-[1,2,4]triazolo[3,4-a]isoquinolin-3-yl)-3-arylprop-2-en-1-one were synthesized, and the structure of their molecules was confirmed through NMR spectroscopy. The antitumor activity of these newly synthesized chalcone derivatives was tested on mouse (Luc-4T1) and human (MDA-MB-231) breast cancer cell lines. The antiproliferative effect was evaluated through SRB screening and the MTT assay after 48 h of treatment at different concentrations. Interestingly, among the tested chalcone derivatives, chalcone analogues with a methoxy group were found to have significant anticancer activity and displayed gradient-dependent inhibition against breast cancer cell proliferation. The anticancer properties of these unique analogues were examined further by cytometric analysis of the cell cycle, quantitative PCR, and the caspases-Glo 3/7 assay. Chalcone methoxy derivatives showed the capability of cell cycle arrest and increased Bax/Bcl2 mRNA ratios as well as caspases 3/7 activity. The molecular docking analysis suggests that these chalcone methoxy derivatives may inhibit anti-apoptotic proteins, particularly cIAP1, BCL2, and EGFRK proteins. In conclusion, our findings confirm that chalcone methoxy derivatives could be considered to be potent drug candidates against breast cancer.

Abstract 5598: Microfluidic isolation and capture of circulating tumor cells and clusters from mouse blood

Abstract Background. Circulating tumor cells (CTCs) and their clusters have been regarded as potential targets for therapies and subjects for identifying certain mechanisms of cancer metastasis. Mouse models have been especially useful tools to investigate the process of metastasis, since they are easier to control and are less variable compared to clinical patient samples. Therefore, it is of interest to extract and study CTCs and CTC clusters from such mouse models. However, most isolation systems are designed and tested for human blood and cell lines. Sized-based methods of CTC isolation have been optimized for human cell lines, but mouse CTCs are generally smaller in size (i.e., ~14 µm EO771 breast cancer cells). Therefore, mouse cell separation necessitates a varied protocol for CTC isolation. We used mouse blood and EO771, a mouse breast cancer cell line, to demonstrate the isolation of mouse CTCs from mouse blood using a sized-based microfluidic device. We also validated the presence of endogenous CTCs and CTC-neutrophil clusters in a tumor bearing mouse model. Methods. Our microfluidic device employs inertial migration in a straight channel segment (150 µm × 50 µm × 24 mm) to isolate CTCs from blood samples. We spiked 100 and 1000 ZsGreen-expressing EO771 cells in 5 × diluted blood containing tdTomato-expressing neutrophils, easily distinguishing CTCs from the red-colored neutrophils. To enumerate the recovered cells, we used Cytospin to concentrate the cells into a glass slide. Lastly, we used hydrodynamic traps to capture CTC clusters from tumor-bearing mouse blood. The blood was extracted from the posterior vena cava of the mice at the endpoint experiment. Results. We achieved an overall recovery rate of 22-29%, which combines the recovery of the isolation device and the Cytospin. Here, we enumerate cells only after their extraction from the collection tube outside of the isolation device. Other methods with higher capture efficiency enumerate the recovered CTCs before they are harvested from the isolation device which is not always representative of the number of cells that are analyzed. We were also able to discover endogenous CTC-neutrophil clusters from tumor-bearing mice which we distinguished apart with anti-EpCAM staining of CTCs (green) and anti-Ly6G (red) staining of neutrophils. Conclusion. We demonstrated and optimized a procedure for isolating mouse-derived CTCs from naïve mice blood and CTC-neutrophil clusters from tumor-bearing mice with our microfluidic isolation devices, as opposed to the isolation of human CTCs as in other sized-based isolation systems. In future studies, we aim to continue to use our microfluidic devices to capture and study CTC and CTC clusters. Citation Format: Celine Macaraniag, Jian Zhou, Ian Papautsky, Jing Li, William Putzbach, Nissim Hay. Microfluidic isolation and capture of circulating tumor cells and clusters from mouse blood. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5598.

Abstract 586: Development of a novel pan-chemokine receptor inhibitor as therapeutic for breast cancer

Abstract The interaction of chemokines and their cognate receptors (CCRs) forms a key signaling network dedicated to promoting an inflammatory tumor microenvironment (TME), leukocyte recruitment and activation, angiogenesis, and growth in all stages of various cancers. Aberrant expressions of chemokines and CCRs are found to correlate with immunosuppression, metastasis, drug resistance, and poor prognosis in breast cancer patients. Several specific inhibitors for chemokines or CCRs are currently tested in clinical trials of multiple cancers. To date, no pan-CCR inhibitor has been developed as a cancer therapy, which is expected to have more clinical advantages than the agent with specificity for a single CCR. We collaborated with Dr. Filip Fratev's team (Micar21, Bulgaria) to develop potent pan-CCR inhibitors for breast cancer. By performing a virtual screen of 4 million compounds based on the analyses of Pharmacophore-based Docking, Induced-fit Docking, Molecular Dynamics, and Metadynamics, a novel pan-CCR inhibitor, SSFF-02, was identified, which showed strong inhibitory activities against CCR1, 3, 5. We tested SSFF-02 in xenografts bearing tumors derived from mouse breast cancer cell line 4T1. The treatments of SSFF-02 (5, 30, and 150 mg/kg) inhibited tumor growth on day 31 and significantly diminished the metastasis in the lung, liver, and ribs. We further examined the levels of serum INF-γ and 26 crucial cytokines and chemokines in the mice treated with SSFF-02 and the control group by enzyme-linked immunosorbent assay (ELISA) and chemokine array assay (RayBiotech). The SSFF-02-treated mice showed significant decreases in serum INF-γ and increases in CXCL5, CCL9, and CCL12. These data suggest that SSFF-02 can module the TME in breast cancer xenografts. The total mRNA of tumors collected from 4T1 xenografts have been harvested and are currently subjected to RNA sequencing (RNAseq) to characterize the chemokine-associated signature of breast cancer in response to SSFF-02. Furthermore, our MTT assays in human triple-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468, Hs578T, and BT-20) and the hormone-dependent MCF7 cell line showed that 25 mM SSFF-02 significantly decreased over 50% of cell viabilities in all tested lines. Matrigel invasion assay demonstrated that the treatment of 5 or 10 mM SSFF-02 inhibited more than 60% of cancer cell migration in all lines. Our studies show that SSFF-02 is a promising targeted therapeutic and immune modulator for metastatic breast cancer. This study provides a foundation to pre-clinically develop SSFF-02 as therapeutics for breast cancer and other inflammatory cancers, such as lung, colorectal, and melanoma, with documented aberrant chemokine signaling. Citation Format: Mariana Grigoruta, Filip Fratev, Yong Qin. Development of a novel pan-chemokine receptor inhibitor as therapeutic for breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 586.

Abstract 2785: Carbidopa/α-methyltryptophan as an ideal combination therapy for breast cancer

Abstract Background/Hypothesis: The amino acid transporter SLC6A14 is upregulated in estrogen receptor (ER)-positive breast cancer, a subtype that represents 75-80% of all breast cancers. Obesity/overweight increases the risk of breast cancer due to estrogen generated from fat cells that fuel the growth of the ER-positive breast cancer. Previous studies from our lab have demonstrated that blockade of the amino acid transporter SLC6A14 by the small molecule α-methyltryptophan reduces the growth of ER-positive breast cancer in mouse xenografts of human breast cancer cells and in a spontaneous mouse model of ER-positive breast cancer. We have also discovered that α-methyltryptophan functions as a potent weight-loss agent. We believe that this compound would be effective for the treatment of ER-positive breast cancer, especially in obese/overweight women. α-methyltryptophan is metabolized by aromatic amino acid decarboxylase. Therefore, combination of this compound with carbidopa, a well-known inhibitor of this enzyme, might potentiate the anticancer efficacy of α-methyltryptophan. We tested this idea in the present study using a syngeneic mouse model of ER-positive breast cancer using the mouse breast cancer cell line AT3. Results: A syngeneic tumor-cell transplant mouse model was used for ER-positive breast cancer. AT3 cells were injected into the mammary fat pad of C57BL/6 mice. This allows to monitor the growth of AT3 tumor growth in an immunologically competent mammary tissue environment. Drug treatment in drinking water was started after tumor growth was noticeable (100 mm3). There were four groups: (i) control; (ii) α-methyltryptophan at 0.5 mg/ml; (iii) carbidopa at 0.25 mg/ml; (iv) α-methyltryptophan at 0.5 mg/ml + carbidopa at 0.25 mg/ml. Tumor growth was monitored every three days. The mice were killed after 4 weeks of treatment. Tumors were excised and weighed. These studies showed that α-methyltryptophan and carbidopa were independently effective in reducing the tumor (48% reduction, p&amp;lt;0.05 for α-methyltryptophan; 50% reduction, p&amp;lt;0.05 for carbidopa), but the combination was the most effective (92% reduction, p&amp;lt;0.01). These effects were evident irrespective of whether the monitored parameter was tumor weight or tumor volume. Conclusion: The combination treatment significantly decreased the tumor size and volume compared to carbidopa and α-methyltryptophan alone. We do not know yet if the improved efficacy of the combination of the two drugs is due to a simple additive effect or due to a synergistic effect. Studies have shown that carbidopa promotes degradation of ER, indicating that carbidopa has its own independent anticancer effect that is relevant to ER-positive breast cancer. Further studies are needed to differentiate between the effect of carbidopa as an independent anticancer agent and its ability to potentiate the anticancer efficacy of α-methyltryptophan by preventing its metabolic degradation. Citation Format: Sirin Falconi, Sabarish Ramachandran, Vadivel Ganapathy. Carbidopa/α-methyltryptophan as an ideal combination therapy for breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2785.

Abstract 3913: Genomic loss of UQCR11 creates therapeutic vulnerability in triple-negative breast cancer

Abstract Recurrent loss-of-function deletions are prevalent genomic alterations in tumors; nonetheless, these deletions occasionally create conditional therapeutic vulnerabilities in tumors. Our previous study used The Cancer Genome Atlas High-Grade Serous Ovarian Carcinoma (TCGA-HGSOC) dataset to identify the 19p13.3 locus as the most pervasive deletion in the high-grade serous ovarian carcinoma (HGSOC). Subsequently, the machine learning integrated-precision oncology platform identified that the loss of UQCR11, a gene located within the chromosome 19p13.3 locus, created a therapeutic collateral target, the MTHFD2. Furthermore, pharmacological inhibition of MTHFD2 in the UQCR11-deleted cells led to complete tumor remission in our in vivo mouse model (Nature metabolism, 2022). Encouraged by the promising anti-tumor effect of the MTHFD2 inhibitor, we extended our research to other tumor types. By analyzing the TGCA datasets, we found that UQCR11 deletion was a common occurrence among humans' most prevalent solid tumors, especially in pan breast and lung cancer. Within the pan breast cancer dataset, we found that over 50% of patients with triple-negative breast cancer (TNBC), the most difficult-to-treat breast cancer subtype in women, displayed either heterozygous or homozygous deletions in the UQCR11 gene. Moreover, the TGCA-TNBC dataset analysis revealed that the genomic copy loss of UQCR11 positively correlates with the deceased mRNA expression of the gene, bringing our interest to validate the collateral lethal targets in this breast cancer subtype. We first analyzed The Cancer Cell Line Encyclopedia (CCLE) database and further explored the correlation between the copy number alteration and mRNA expression of UQCR11 in several breast cancer cell lines. Then, we selected seven human TNBC cells with copy number neutral or deletion UQCR11 and validated the copy number and gene expression of UQCR11 and MTHFD2 using qPCR and western blot. We next treated these cells with a selective MTHFD2 inhibitor, DS18561882, and found that UQCR11-deleted cells showed a more favorable response than the UQCR11-intact cells. Finally, to test the therapeutic potential of targeting MTHFD2-null tumors in vivo, we generated isogenic mouse breast cancer cell lines using shRNA-mediated UQCR11 knockdown. Consistent with our previous findings, the MTHFD2 expression level was upregulated upon UQCR11 knockdown, suggesting a compensation function of MTHFD2 in UQCR11-deleted cells. Furthermore, the UQCR11 knocked-down cells showed better responses to MTHFD2 inhibitor treatment than the parental control cells. In summary, our study provides evidence that targeting MTHFD2 holds strong therapeutic potential in over 50% of the TNBC patients with UQCR11 deletion and highlights the broad efficacy of targeting MTHFD2 for individualized therapeutic strategy in different tumor types co-occurring with UQCR11 deletion. Citation Format: Samantha Sharma, Tao Yu, Abhinav Achreja, Xinna Zhang, Deepak Nagrath, Xiongbin Lu. Genomic loss of UQCR11 creates therapeutic vulnerability in triple-negative breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3913.

Abstract 2517: Optimizing breast cancer therapy by inhibiting the adenosine receptor and oxygen consumption

Abstract Immune checkpoint blockade has shown remarkable promise in melanoma and other tumor types. However, a large proportion of patients do not respond or develop acquired resistance. Tumors can activate multiple checkpoints and immunosuppressive pathways to evade immune surveillance, which are likely a cause of treatment failure. Therefore, inhibition of multiple checkpoints may be necessary for maximal efficacy of immunotherapies. The adenosine A2 receptor (A2aR), was shown to function as an immune checkpoint. Its blockade inhibited tumor growth and metastases and synergized with other checkpoint inhibitors. We propose that using AZD4635, a potent and selective A2aR antagonist, will enhance the therapeutic efficacy of anti-PD-1/PD-L1. In addition, we propose that blocking tumor oxygen consumption using deferiprone (DFP), phenformin (Phen) and metformin (Met) will further enhance A2aR and PD-1/PD-L1 blockade efficacy. We evaluated the effects of MET, Phen, DFP, and AZD4635 on tumor cells, T cell, and macrophage proliferation and effector function in vitro. IC50 measurements showed that T cells are the most sensitive to inhibition by these drugs, while 4T1 tumor cells appear to be the least sensitive. Naïve mouse T cells were activated with anti-CD3 and anti-CD28 coated dynabeads in the presence of titrating doses of all four drugs for 72 hours then analyzed by flow cytometry for proliferation and activation status. AZD4635 and DFP increased the expression of the activation marker CD25 (IL2Ra) on CD8 T cells. In addition, AZD4635 also increased expression of Granzyme B on both CD4 and CD8 T cells. Met appears to have little to no effect on T cell proliferation activation. We utilized the Seahorse XF Mito Stress Test assay to measure the mitochondrial respiratory activity of T cells in the presence these drugs. T cells were activated with dynabeads for 72 hours and then incubated with titrating doses of the drugs overnight before running on the Seahorse assay. DFP and Phen had the strongest effect on Maximal Respiration and Spare Respiratory Capacity at dilutions of 1uM followed by 16uM. A similar, albeit less profound effect was observed in groups treated with Met and AZD4635. Lastly, we obtained and generated two mouse breast cancer cell lines (4T1 and E0771) bearing HIF-1 reporter systems. We treated 4T1-HRE and E0771 cells with increasing doses of cobalt chloride (CoCl2) to chemically mimic hypoxia by inducing HIF1a expression. Both 4T1-HRE and E0771-HRE demonstrated increased luciferase activity that correlated with increasing doses of CoCl2 in vitro. Future experiments will focus on characterizing the adenosine pathway in vivo in 4T1-HRE and E0771-HRE tumors and examine how drugs that target the adenosine A2AaR receptor (AZD4635), and oxygen consumption (DFP, Phen and Met) influence tumor oxygen consumption in vivo as well as the activation states of immune cells in the tumor microenvironment. Citation Format: Sadna Budhu, Mayuresh Mane, Mamadou A. Bah, Juan Zurita, Inna Serganova, Soe Min, Anais Assouvie, Jedd D. Wolchok, Jason Koutcher, Vladimir Ponomarev, Taha Merghoub. Optimizing breast cancer therapy by inhibiting the adenosine receptor and oxygen consumption [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2517.

Abstract 3604: Epithelial Caveolin-1 regulates lung metastasis in advanced stages of breast cancer

Abstract Organotypic metastasis is a well-orchestrated sequence of events that takes place in tumors. The interplay between secretory factors from the primary tumor and the microenvironment of a distant organ determines the fate of metastasis. A significant concern in effective triple-negative breast cancer (TNBC) treatment is lung metastasis. Caveolin-1 (Cav-1), a lipid raft protein, is an important component of the metastatic interplay and is reported to be upregulated in the advanced metastatic stage of breast cancer. Although Cav-1 has been shown to have oncogenic potential, its role in TNBC lung metastasis is not fully understood. Our data illustrates, knocking out Cav-1 in the 4T1 cells (a mouse breast cancer cell line) affects cell morphology, extracellular vesicle formation, and MMP release in vitro. Furthermore, a wound healing/cell migration assay showed reduced migration in Cav-1 KO 4T1 cells when compared to control 4T1 cells, and these findings were validated using Cav-1 siRNA. For investigating the metastatic role, we injected Cav-1 KO 4T1 and control 4T1 cells into the mammary fat pad of female BALB/c mice, a syngeneic mouse model of triple-negative breast cancer. We found no evidence of lung metastasis in Cav-1 KO 4T1 injected mice when compared to control 4T1 injected mice. This was further supported by mRNA profiling of the tumor. We observed 21 epithelial cell migration genes that are differentially expressed in Cav-1 KO tumors when compared to WT tumors. Correlation analysis from Breast Cancer Gene-Expression Miner v4.8 (bc-GenExMiner), a human database, showed a significant correlation between Cav-1 expression and several of the 21 epithelial cell migration genes, along with integrin 3 alpha (ITGα3). Interestingly, in silico protein docking predicted an interaction between Cav-1 and ITGα3, which was later confirmed by co-immunoprecipitation. The role of ITGα3 in metastasis was further corroborated by cell migration assay, which was affected when siRNA was used to silence ITGα3 gene expression. Collectively, these findings provide evidence that Cav-1 KO, along with ITGa3, plays an important role in distant lung metastasis in the 4T1 tumor-induced mice model. Citation Format: Dhirendra Pratap Singh, Rashmi Pathak, Abhishek Pandit, Philip J. Ebenezer, Nithya Jambunathan, Sanjay Kumar, Alexander Du Plooy, Joseph Francis, Mary E. White. Epithelial Caveolin-1 regulates lung metastasis in advanced stages of breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3604.

Design of novel polyurethane-based ionene nanocarriers for cancer therapy: Synthesis, in-vitro, and in-vivo studies

New strategies for constructing versatile nanocarriers are needed for cancer therapy to overcome the multiple challenges of targeted delivery. This work explores the advantages of polyurethane with main-chain quaternary ammonium salt moieties (ionene) as a novel carrier for targeted drug delivery. We have developed a novel cationic soybean oil-based polyurethane ionene nanocarrier (CPUI) that can act as an effective anticancer agent and efficiently deliver the anticancer drug 5-fluorouracil (5FU). We also report a potential anticancer drug delivery system targeting the folate receptor. In vitro experiments with blank CPUI carriers on the 4T1 (mouse breast cancer cell line) and the NIH-3T3 (mouse fibroblast cell line) revealed high cytotoxicity for the cancer cells but only low cytotoxicity for the normal fibroblast cells. The CPUI nanoparticles were readily loaded with 5FU (5FU-CPUI) in water using electrostatic interactions between the cationic quaternary ammonium groups of ionene and the anionic 5FU. The in vivo study in mice with tumors showed that the blank CPUI carriers significantly inhibited tumor growth, even more than the free drug (5FU). The inhibitory effect on tumor growth was slightly enhanced when the carriers were loaded with 5FU. The prepared nanoparticles had a high loading capacity of 41.8 %. Further enhancement of the inhibitory effect was observed when folic acid (FA) was added as a targeting moiety to the system via ion exchange with the bromine counterion of the quaternary ammonium moieties. The results suggest that the efficacy of FA-CPUI-5FU nanoparticles as vehicles for drug delivery can be enhanced via folate receptor (FR) mediated endocytosis in 4T1 cells and these novel nanocarriers may provide a potential platform for effective targeted drug delivery to tumor tissue and breast cancer therapy in the clinic.

Abstract B022: pH-low insertion peptide detects lactic acidosis contributing metastatic niche formation in lungs

Abstract Background: Pre-metastatic niche refers to the environment prepared for efficient colonization of disseminated cancer cells in specific organs. Primary tumor secretome facilitates pre-metastatic niche through a series of molecular and cellular changes. This study focuses on interstitial pH in organs with pre-metastatic niche because dysregulated pH homeostasis plays a well-established role in tumor metastasis. In primary tumor, extracellular acidification directly promotes invasion/migration of cancer cells for extravasation. Meanwhile, little is known regarding interstitial pH in organs that are distant from primary tumor. Methods: BALB/c mice were subcutaneously inoculated with 4T1.2 cells or 66cl4 cells (mouse breast cancer cell lines) with resistance against 6-thioguanine. Absence of metastatic cells in their lungs was confirmed by long-termed culture of dissociated pulmonary cells in presence of 6-thioguanine; it defines Day 21 from inoculation as a pre-metastatic phase. Alternatively, 4T1.2 cell-conditioned media (4T1.2-CM) or control media was intraperitoneally administered every day for 21 consecutive days. pH-low insertion peptide (pHLIP® peptide) stably labels cells exposed to acidic conditions, which is feasible to evaluate interstitial acidity in each organ. Alexa Fluor® 750-labelled pHLIP® peptide was injected into mice, and fluorescent signals in harvested organs were detected by IVIS Lumina XRMS Series III. For a metastasis model, mice first received 4T1.2-CM or in combination with sodium oxamate (a lactate dehydrogenase inhibitor) followed by an intravenous injection of 66cl4 cells expressing luciferase. Results: At the pre-metastatic phase, 4T1.2 tumor-bearing mice showed great accumulation of pHLIP® peptide and high amounts of lactate in their lungs. By contrast, there was no significant fluorescence from pHLIP® peptide in lungs of low-metastatic 66cl4 cell-inoculated mice. Notable accumulation of pHLIP® peptide was also observed in the lungs of 4T1.2-CM-given mice. Intravenously injected 66cl4 cells colonized more aggressively in the acidic lungs than in control ones. Suppression of the lung acidification by sodium oxamate attenuated the metastatic burden, which significantly improved survival of the mice. In the acidic lungs, pHLIP® peptide was localized in alveolar type II (AT2) cells expressing surfactant protein C. Sorted AT2 cells from the acidic lungs strongly expressed glycolysis-related proteins. Conclusions: This study using pHLIP® peptide indicates a highly glycolytic activity with increased lactic acid in the lungs of 4T1.2 tumor-bearing mice at the pre-metastatic phase. Mechanistically, soluble factors derived from 4T1.2 cells could transform AT2 cells into acidic cells, which might provide metastasis-promoting function. This study will shed light on understanding the whole picture of pre-metastatic niche and give scientific insights to develop novel diagnosis and therapy for tumor metastasis. Citation Format: Toma Matsui, Yuki Toda, Anna Mosnikova, Oleg A. Andreev, Shigekuni Hosogi, Yana K. Reshetnyak, Eishi Ashihara. pH-low insertion peptide detects lactic acidosis contributing metastatic niche formation in lungs [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B022.

Gaseous nitric oxide tumor ablation induces an anti-tumor abscopal effect

BackgroundIn-situ tumor ablation provides the immune system with the appropriate antigens to induce anti-tumor immunity. Here, we present an innovative technique for generating anti-tumor immunity by delivering exogenous ultra-high concentration (> 10,000 ppm) gaseous nitric oxide (UHCgNO) intratumorally.MethodsThe capability of UHCgNO to induce apoptosis was tested in vitro in mouse colon (CT26), breast (4T1) and Lewis lung carcinoma (LLC-1) cancer cell lines. In vivo, UHCgNO was studied by treating CT26 tumor-bearing mice in-situ and assessing the immune response using a Challenge assay.ResultsExposing CT26, 4T1 and LLC-1 cell lines to UHCgNO for 10 s–2.5 min induced cellular apoptosis 24 h after exposure. Treating CT26 tumors in-situ with UHCgNO followed by surgical resection 14 days later resulted in a significant secondary anti-tumor effect in vivo. 100% of tumor-bearing mice treated with 50,000 ppm UHCgNO and 64% of mice treated with 20,000 ppm UHCgNO rejected a second tumor inoculation, compared to 0% in the naive control for 70 days. Additionally, more dendrocytes infiltrated the tumor 14 days post UHCgNO treatment versus the nitrogen control. Moreover, T-cell penetration into the primary tumor was observed in a dose-dependent manner. Systemic increases in T- and B-cells were seen in UHCgNO-treated mice compared to nitrogen control. Furthermore, polymorphonuclear-myeloid-derived suppressor cells were downregulated in the spleen in the UHCgNO-treated groups.ConclusionsTaken together, our data demonstrate that UHCgNO followed by the surgical removal of the primary tumor 14 days later induces a strong and potent anti-tumor response.