4T1 Murine Breast Cancer Cells Research Articles

The Proapoptotic Action of Pyrrolidinedione-Thiazolidinone Hybrids towards Human Breast Carcinoma Cells Does Not Depend on Their Genotype.

The development of new, effective agents for the treatment of breast cancer remains a high-priority task in oncology. A strategy of treatment for this pathology depends significantly on the genotype and phenotype of human breast cancer cells. We aimed to investigate the antitumor activity of new pyrrolidinedione-thiazolidinone hybrid molecules Les-6287, Les-6294, and Les-6328 towards different types of human breast cancer cells of MDA-MB-231, MCF-7, T-47D, and HCC1954 lines and murine breast cancer 4T1 cells by using the MTT, clonogenic and [3H]-Thymidine incorporation assays, flow cytometry, ELISA, and qPCR. The studied hybrids possessed toxicity towards the mentioned tumor cells, with the IC50 ranging from 1.37 to 21.85 µM. Simultaneously, these derivatives showed low toxicity towards the pseudonormal human breast epithelial cells of the MCF-10A line (IC50 > 93.01 µM). Les-6287 at 1 µM fully inhibited the formation of colonies of the MCF-7, MDA-MB-231, and HCC1954 cells, while Les-6294 and Les-6328 did that at 2.5 and 5 µM, respectively. Les-6287 suppressed DNA biosynthesis in the MCF-7, MDA-MB-231, and HCC1954 cells. At the same time, such an effect on the MCF-10A cells was significantly lower. Les-6287 induces apoptosis using extrinsic and intrinsic pathways via a decrease in the mitochondrial membrane potential, increasing the activity of caspases 3/7, 8, 9, and 10 in all immunohistochemically different human breast cancer cells. Les-6287 decreased the concentration of the metastasis- and invasion-related proteins MMP-2, MMP-9, and ICAM-1. It did not induce autophagy in treated cells. In conclusion, the results of our study suggest that the synthesized hybrid pyrrolidinedione-thiazolidinones might be promising agents for treating breast tumors of different types.

RNA editing enzyme ADAR2 regulates P-glycoprotein expression in murine breast cancer cells through the circRNA-miRNA pathway

Among the various RNA modifications, adenosine-to-inosine RNA editing, catalyzed by adenosine deaminase acting on RNA (ADAR) family, ADAR1 and ADAR2, is the most common nucleotide conversion in mammalian cells. The pathological relevance of ADAR expression has been highlighted in recent human genetic studies. Low expression of the ADAR2 gene is correlated with a poor prognosis in breast cancer patients, but the underlying mechanism remains enigmatic. In this study, we constructed Adar2-knockdown (Adar2-KD) murine breast cancer 4T1 cells and observed their reduced susceptibility to chemotherapeutic drug doxorubicin. Downregulation of ADAR2 induced the expression of P-glycoprotein (P-gp), leading to a reduction in the intracellular accumulation of doxorubicin. The upregulation of P-gp occurred at the post-transcriptional level due to the decreased miR-195a-3p function. The search for the underlying cause of the induction of P-gp expression in Adar2-KD 4T1 cells led to the identification of circular RNA (circRNA) circHif1a as a sponge for miR-195a-3p. The enhanced expression of circHif1a inhibited miR-195a-3p function, resulting in the upregulation of P-gp expression. These results suggest that ADAR2 acts as a suppressor of circHif1a biogenesis and then allows miR-195a-3p to interfere with P-gp translation. Our findings may help to improve drug efficacy by clarifying the mechanism of chemoresistance in breast cancer.

A recombinant adeno-associated virus expressing secretory TGF-β type Ⅱ receptor inhibits triple-negative murine breast cancer 4T1 cell proliferation and lung metastasis in mice

To investigate the effects of an adeno-associated virus (AAV2) vector expressing secretory transforming growth factor-β (TGF-β) type Ⅱ receptor (sTβRⅡ) extracellular domain-IgG2a Fc fusion protein (sTβRⅡ-Fc) on proliferation and migration of triple-negative murine breast cancer 4T1 cells in mice. The pAAV-sTβRⅡ-Fc vector expressing sTβRⅡ-Fc fusion protein constructed by molecular cloning, the capsid protein-expressing vector pAAV2 and the helper vector were co-transfected into HEK 293T cells to prepare the recombinant AAV2-sTβRⅡ virus, which was purified by density gradient centrifugation with iodixanol. Western blotting was used to examine the effects of AAV-sTβRⅡ virus on Smad2/3 phosphorylation in 4T1 cells and on expression levels of E-cadherin, vimentin and p-Smad2/3 in 4T1 cell xenografts in mice. BALB/c mice bearing subcutaneous xenografts of luciferase-expressing 4T1 cells received intravenous injections of AAV-sTβRⅡ virus, AAV-GFP virus or PBS (n=6) through the tail vein, and the proliferation and migration of 4T1 cells were analyzed with in vivo imaging. Ki67 expression in the tumor tissues and sTβRⅡ protein expressions in mouse livers were detected with immunohistochemistry and immunofluorescence staining, and tumor metastases in the vital organs were examined with HE staining. The recombinant pAAV-sTβRⅡ-Fc vector successfully expressed sTβRⅡ in HEK 293T cells. Infection with AAV2-sTβRⅡ virus significantly reduced TGF-β1-induced Smad2/3 phosphorylation in 4T1 cells and effectively inhibited proliferation and lung metastasis of 4T1 xenografts in mice (P<0.05). In the tumor-bearing mice, intravenous injection of AAV-sTβRⅡ virus significantly increased E-cadherin expression, reduced vimentin and Ki67 protein expressions and Smad2/3 phosphorylation level in the tumor tissues (P<0.05 or 0.01), and induced liver-specific sTβRⅡ expression without causing body weight loss or heart, liver, spleen or kidney pathologies. The recombinant AVV2 vector encoding sTβRⅡ extracellular domain is capable of blocking the TGF-β signaling pathway to inhibit the proliferation and lung metastasis of 4T1 cells in mice.

Establishing and Characterizing Chemotherapy-Induced Immunological Dormant Tumor Cell Lines.

Chemotherapy, together with radiotherapy, targeted therapies, and immunotherapy, is the main option to treat cancer patients in neoadjuvant/adjuvant setting to reduce the risk of disease progression and metastasis formation from disseminated tumor cells. Cancer cells that survived chemotherapy treatment may emerge with novel characteristics, one of which is the ability to stimulate the native and adaptive immune systems. Models allowing the characterization of chemotherapy-induced tumor cell plasticity and induction of immune response or adaptation are needed to identify novel mechanisms and devise novel therapeutic strategies to prevent relapses. Here we describe a protocol for selecting chemotherapy-resistant cancer cells and testing the in vivo effects on the local and systemic immune responses. While originally developed to characterize the effects of methotrexate and doxorubicin on murine 4T1 breast cancer cells and the relative immune response, the method can be broadened to other chemotherapies and syngeneic cancer models.

Interferon-tau (IFN-τ) Has Antiproliferative Effects, Induces Apoptosis, and Inhibits Tumor Growth in a Triple-negative Breast Cancer Murine Tumor Model.

Resistant triple-negative breast cancer (TNBC) is a subtype of this disease that is resistant to conventional chemotherapy agents. IFN-τ is a cytokine that has recently been shown to have immunoregulatory and antitumor effects. The present study aimed to examine the antiproliferative and apoptosis effects of IFN-τ in breast cancer cells and the antitumor effect in a murine tumor model of TNBC. Murine breast cancer 4T1 cells were cultured and treated with ovine IFN-τ and through MTT and Caspase-Glo 3/7 assays, viability and cell death were determined. In addition, the antitumor effect of IFN-τ was determined in a murine tumor model of TNBC. Ovine IFN-τ showed a concentration-dependent antiproliferative effect on 4T1 murine breast cancer cells. Also, treatment of 4T1 cells with IFN-τ induced the activation of caspase 3 and 7, which is indicative of apoptotic cell death. Moreover, we detected an increase in the expression of type I interferon receptor (IFNAR1/2) in cells treated with IFN-. The intratumoral application of IFN-τ in mice inhibited tumor growth compared to the control non-treated group, and the effect was associated with the increased expression of GM-CSF. Ovine IFN-τ may be an effective immunotherapeutic cytokine for the treatment of TNBC.

Production of Soluble Murine TRAILs in Escherichia coli with Zn2+ Supplementation.

Accumulating evidence has demonstrated the immunomodulatory effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in rheumatoid arthritis and the tumor microenvironment, besides its known capacity of specifically inducing the apoptosis of cancer cells. Mice are common available animal models for studying the roles of TRAIL. However, mice express only a single TRAIL receptor (mTRAILR) with an intracellular death domain, in contrast to the two TRAIL receptors (TRAILR1 and TRAILR2) in humans. Moreover, human TRAIL binds weakly to mTRAILR, whereas mouse TRAIL has a high affinity for human TRAIL-Rs. Therefore, we considered that murine TRAIL would be more suitable than human TRAIL for exploring the immunoregulatory effect of TRAIL in immunocompetent mice or when using mouse cells as the target. To our knowledge, the detailed method for the production of recombinant murine TRAIL has not been reported. In this study, we aimed to design and express two soluble forms of murine TRAIL and verify the properties of the protein. Recombinant murine TRAILs were expressed in Escherichia coli BL21 (DE3, and Nichelating affinity chromatography was used for protein purification. SDS-PAGE, GDS-PAGE and HPLC were applied to analyze the protein structure. The cytotoxicity of our purified murine TRAILs was evaluated in the TRAIL-sensitive human breast cancer ZR-75-30 cells and murine breast cancer 4T1 cells. Finally, validation of the tumor-killing ability of the murine protein in vivo. Two soluble forms of murine TRAILs (mT_N99 and mT_N188) were purified and demonstrated with high purity and trimeric structure. In addition, Zn2+ supplement was essential to produce soluble murine TRAILs in E.coli BL21 (DE3). The two purified soluble mTRAILs showed similar cytotoxicity to cancer cells, moreover, mT_N99 also showed a good anti-tumor effect in vivo and is more suitable for the treatment of murine tumor models. A production approach for recombinant murine TRAIL was determined, which covered the design of shortened forms, expression, purification and characterization.

Discovery of 3-Aminopyrazole Derivatives as New Potent and Orally Bioavailable AXL Inhibitors.

The receptor tyrosine kinase AXL is a promising target for anticancer drug discovery. Herein, we describe the discovery of 3-aminopyrazole derivatives as new potent and selective AXL kinase inhibitors. One of the representative compounds, 6li, potently inhibited AXL enzymatic activity with an IC50 value of 1.6 nM, and tightly bound with AXL protein with a Kd value of 0.26 nM, while was obviously less potent against most of the 403 wild-type kinases evaluated. Cell-based assays demonstrated that compound 6li potently inhibited AXL signaling, suppressed Ba/F3-TEL-AXL cell proliferation, reversed TGF-β1-induced epithelial-mesenchymal transition, and dose-dependently impeded cancer cell migration and invasion. Compound 6li also showed reasonable pharmacokinetic properties in rats and exhibited significant in vivo antitumor efficacy in a xenograft model of highly metastatic murine breast cancer 4T1 cells. Taken together, this study provides a new potent and selective AXL inhibitor for further anticancer drug discovery.

Chronic intermittent hypoxia, a hallmark of obstructive sleep apnea, promotes 4T1 breast cancer development through endothelin-1 receptors

The association between obstructive sleep apnea (OSA) and cancer is still debated and data are scarce regarding the link between OSA and breast cancer progression. Since conclusive epidemiological studies require large sample sizes and sufficient duration of exposure before incident cancer occurrence, basic science studies represent the most promising approach to appropriately address the topic. Here we assessed the impact of intermittent hypoxia (IH), the major hallmark of OSA, on the development of breast cancer and explored the specific involvement of the endothelin signaling pathway. Original in vitro and in vivo models were used where 3D-spheroids or cultures of murine 4T1 breast cancer cells were submitted to IH cycles, and nude NMRI mice, orthotopically implanted with 4T1 cells, were submitted to chronic IH exposure before and after implantation. The role of the endothelin-1 in promoting cancer cell development was investigated using the dual endothelin receptor antagonist, macitentan. In vitro exposure to IH significantly increased 4T1 cell proliferation and migration. Meta-analysis of 4 independent in vivo experiments showed that chronic IH exposure promoted tumor growth, assessed by caliper measurement (overall standardized mean difference: 1.00 [0.45–1.55], p < 0.001), bioluminescence imaging (1.65 [0.59–2.71]; p < 0.01) and tumor weight (0.86 [0.31–1.41], p < 0.01), and enhanced metastatic pulmonary expansion (0.77 [0.12–1.42]; p = 0.01). Both in vitro and in vivo tumor-promoting effects of IH were reversed by macitentan. Overall, these findings demonstrate that chronic intermittent hypoxia exposure promotes breast cancer growth and malignancy and that dual endothelin receptor blockade prevents intermittent hypoxia-induced tumor development.

Increased lung metastasis with sevoflurane in breast cancer surgery is associated with VEGF and elevated vascular permeability by VEGF in a mouse model of human breast cancer.

e13060 Background: An on-going controversy is whether general anesthetics used in cancer surgery substantially influence the outcome of cancer patients. Thousands of cancer patients undergo surgery with hope of cure. However, postoperative metastasis remains a deadly disease that affects millions of lives, even with all the recent therapeutic advancements. Anesthesia is required for surgical resection of solid tumors and we have shown that general anesthetics substantially influences the process of breast cancer lung metastasis through increasing pulmonary permeability in animal models. Our previous study demonstrates that inhaled anesthetic sevoflurane used during resecting primary breast tumors promotes lung metastasis via IL6 pathway in syngeneic model of murine 4T1 breast cancer cells, compared with an intravenous anesthetic propofol. Methods: Xenograft model of human MDA-MB 231 breast cancer of spontaneous metastasis was utilized in this study to evaluate the effect of commonly used general anesthetics including sevoflurane and propofol on breast cancer lung metastasis and explore the potential mechanism. Results: In this study we show that the metastatic promoting effect of sevoflurane is associated with elevated secretion of VEGF and increased vascular permeability in xenograft model of human MDA-MB 231 breast cancer of spontaneous metastasis. Cytokine array profiling reveals that the mice receiving sevoflurane have increased levels of pro-angiogenic factor VEGF in serum and lung tissues. Sevoflurane enhances the expression level of VEGFR on HUVEC cells. Furthermore, sevoflurane disrupts endothelial monolayer in vitro and elevates pulmonary vascular permeability in vivo. The sevoflurane-promoted breast cancer lung metastasis is reversed by DC101, an antibody targeting mouse VEGFR-2. Conclusions: Our results show that the inhaled anesthetic sevoflurane during surgical removal of primary tumor alter the course of metastasis through regulating secretion of VEGF and associated vascular remodeling. The mechanistic study of general anesthetics on cancer cell metastasis establishes the causal link and could provide the potential therapeutic intervention and guide the clinical trials.

No Difference Among Inhaled Anesthetics on the Growth and Metastasis of Murine 4T1 Breast Cancers in a Mouse Model of Spontaneous Metastasis.

Objective: This study evaluates the effect of the commonly used inhaled anesthetics isoflurane, sevoflurane, and desflurane on the viability and migration of murine 4T1 breast cancer cells, the growth, and lung metastasis in a syngenetic model of spontaneous metastasis. Methods: The murine 4T1 breast cancer cells were exposed to isoflurane (2%), sevoflurane (3.6%), or desflurane (10.3%) for 3 h. Cell viability was measured using the MTT assay. The migratory capacity of 4T1 cells was assessed using a scratch assay after 24 h incubation. Female balb/c mice were subjected to orthotopic implantation of 4T1 cells under anesthesia with one of the inhaled anesthetics: 2% isoflurane, 3.6% sevoflurane, or 10.3% desflurane. Subsequently, resection of primary tumors was performed under the identical anesthetic used during implantation for 3 h. Three weeks later, the mice were euthanized to harvest lungs for ex vivo bioluminescent imaging and histological analysis. Blood was collected for serum cytokine assays by ELISA. Results: There was no difference in cell viability among isoflurane, sevoflurane, desflurane, and control groups (n = 180 for each group, P = 0.648). Sevoflurane but not isoflurane or desflurane significantly increased the migration of 4T1 cells compared to the control group (n = 18, P = 0.024). There was no difference in the growth of the orthotopically implanted primary tumors (n = 12 for the isoflurane group, n = 11 for the sevoflurane group, and for the desflurane group, P = 0.879). Surgical dissection of primary tumors in mice under anesthesia with isoflurane, sevoflurane, or desflurane led to no difference in lung metastasis following surgery (P = 0.789). No significant difference was observed among isoflurane, sevoflurane, and desflurane groups in the serum levels of IL-6 (P = 0.284), CCL-1 (P = 0.591), MCP-1 (P = 0.135), and VEGF (P = 0.354). Conclusion: Our study demonstrated that sevoflurane increased the migration of 4T1 breast cancer cells in vitro. Inhaled anesthetics isoflurane, sevoflurane, and desflurane had no difference on the growth of primary tumor and the lung metastasis of 4T1 cells in the mouse model of spontaneous metastasis with surgical removal of primary tumors.

Sonosensitized Aggregation-Induced Emission Dots with Capacities of Immunogenic Cell Death Induction and Multivalent Blocking of Programmed Cell Death-Ligand 1 for Amplified Antitumor Immunotherapy

Sonosensitized Aggregation-Induced Emission Dots with Capacities of Immunogenic Cell Death Induction and Multivalent Blocking of Programmed Cell Death-Ligand 1 for Amplified Antitumor Immunotherapy

RADI-20. Brain metastasis treatment with high energy radiotherapy and Cherenkov radiation-activated phototherapy

Abstract Radiation therapy is a mainstay in the treatment of brain metastasis, yet some tumors are resistant, and elsewhere brain recurrence outside the radiation field is common. Phototherapy using UV light-activated compounds can both kill cancer cells directly and trigger an immune response to extend tumor control. Poor penetration depth of ultraviolet light, however, has limited this treatment to superficial tumors. High-energy photon beams create high energy electrons within the patient which in turn produce Cherenkov radiation in the UV spectrum while traveling through tissue. Given that this Cherenkov radiation is generated deep within the patient and has the ability to activate photosensitive compounds, we therefore developed a platform to test this phenomenon to enhance radiation therapy for brain metastasis. We first tested UV-activated psoralen derivatives in combination with UV light in vitro for activity against murine 4T1 breast cancer cells, and then irradiated an ex vivo organotypic brain slice platform using a high energy linear accelerator to generate Cherenkov radiation. We tested the survival of 4T1 cells expressing fluorescent and bioluminescent reports in the presence and absence of these psoralen compounds in this ex vivo brain metastasis model. 8-methoxypsoralen (8-MOP) and 4’-Aminomethyltrioxsalen hydrochloride (AMT) both showed 365nm UVA light-specific cell killing in vitro. We optimized AMT cell loading (1 hour) and concentrations [1μM] AMT to maximize cytotoxicity. Testing of AMT using the organotypic brain slice platform and high-energy irradiation to generate Cherenkov-UV light demonstrated similar enhanced cell death of 4T1 cells despite high baseline levels of radiation-induced tumor cell kill. Cherenkov radiation-induced photo-activation of AMT improved cell killing in an ex vivo model of breast cancer brain metastasis. This application holds promise for the re-treatment of refractory tumors with high-energy, low dose radiation, and enhanced elsewhere brain metastasis control through activation of the immune system.

Post-transcriptional repression of circadian component CLOCK regulates cancer-stemness in murine breast cancer cells.

Disruption of the circadian clock machinery in cancer cells is implicated in tumor malignancy. Studies on cancer therapy reveal the presence of heterogeneous cells, including breast cancer stem-like cells (BCSCs), in breast tumors. BCSCs are often characterized by high aldehyde dehydrogenase (ALDH) activity, associated with the malignancy of cancers. In this study, we demonstrated the negative regulation of ALDH activity by the major circadian component CLOCK in murine breast cancer 4T1 cells. The expression of CLOCK was repressed in high-ALDH-activity 4T1, and enhancement of CLOCK expression abrogated their stemness properties, such as tumorigenicity and invasive potential. Furthermore, reduced expression of CLOCK in high-ALDH-activity 4T1 was post-transcriptionally regulated by microRNA: miR-182. Knockout of miR-182 restored the expression of CLOCK, resulted in preventing tumor growth. Our findings suggest that increased expression of CLOCK in BCSCs by targeting post-transcriptional regulation overcame stemness-related malignancy and may be a novel strategy for breast cancer treatments.

Glutathione-activated DNA-Au nanomachine as targeted drug delivery platform for imaging-guided combinational cancer therapy

Multimodal therapy is a combination of different therapeutic modalities within one single platform to enhance treatment efficiency, which holds great promise in biomedical research and clinical practice. Herein, a glutathione (GSH)-activated DNA-Au nanomachine with tumor-targeting capacity has been developed for imaging-guided photodynamic/photothermal/chemo combination therapy of murine breast cancer. In this theranostic nanoplatform, G-quadruplex double-stranded DNA (G-dsDNA) containing AS1411 aptamer is served as not only the targeting unit to specifically recognize murine 4T1 breast cancer cells but also the carrier for loading photosensitizer chlorins e6 (Ce6), iron-containing porphyrin hemin and anticancer drug doxorubicin (Dox). In tumor microenvironment, the excessive GSH triggers the rupture of disulfide bridges and results in the dissociation of DNA-Au nanomachine along with the release of Dox for chemotherapy and the aggregation of AuNPs for photothermal therapy (PTT) as well as in situ imaging. Meanwhile, hemin and Ce6 delivered by G-quadruplexes alleviate the tumor hypoxia, which further promotes the generation of reactive oxygen species (ROS) for enhanced photodynamic therapy (PDT). The in vitro and in vivo assays validate that the combination of PDT/PTT/chemotherapy shows a high efficiency for targeted damage of 4T1 cells and inhibition of tumor growth. Therefore, the proposed multifunctional DNA-Au nanomachine provides a powerful and robust theranostic nanoplatform for imaging-guided combinatorial anticancer therapy.

Abstract LT011: Pharmacological blockade of platelet-CysLT1 receptor counteracts platelet protumoral action and prevents breast cancer cell metastasis to bone and lungs

Abstract Metastases are the main cause of death in cancer patients. However, few metastasis-specific targets have been exploited therapeutically. Blood platelets are well known to contribute to the poor survival of patients with advance cancers. Unfortunately, platelets are mandatory for human life due to their paramount function in hemostasis. In order to define new anti-metastasis therapies exhibiting a high specificity for the pathological tumor cell-platelet interactions without abrogating normal platelet functions we developed a high throughput assay that showed that pharmacological blockade of cysteinyl leukotriene receptor 1 (CysLT1) with two already FDA-approved drugs in asthma (Zarfirlukast and Montelukast), dose-dependently inhibited the mitogenic action of human platelets on human MDA-B02/Luc breast cancer cells. Both drugs had no impact on cell proliferation in presence of serum suggesting a specific alteration of platelet function. This effect was specific to CysLT1 as the CysLT2 inhibitor HAMI3379 remained inefficient even at high doses. All human breast cancer cells including MDA-B02/Luc cells used in this study did not express CysLT1 whereas platelets do suggesting that platelet-CysLT1 was the one contributing to the protumoral action of platelets. As described previously cysteinyl leukotriene D4 (LTD4), the strongest agonist of CysLT1, has no direct effect on platelet aggregation but instead potentiates the action of platelet agonists. We next injected MDA-B02/Luc cells into the tail artery of nude mice that received a daily treatment with Zafirlukast (0.4mg/kg per os). After 7 days bone marrow cells were collected and placed in culture in presence of puromycine for 14 day. After staining with crystal violet clones of tumor cells that colonized bone (TCB) were quantified. In these conditions Zafirlulast significantly inhibited by 90% the number of TCB. In order to confirm this anti-metastasis effect we used a syngenic animal model of spontaneous metastasis dissemination of murine 4T1 breast cancer cells after orthotopic xenograph in the mammary gland of BALB/c immunocompetent mice. Animals were treated with Zafirlukast (0.4mg/kg/day, i.p.), the common chemotherapeutic agent Paclitaxel (30mg/kg/2 days, i.p), or both drugs combined starting at day 3 post-tumor cell injections until sacrifice at day 12. Lungs were collected, minced, treated with collagenase and placed in culture for 14 days in presence of 6-Thioguanine. After staining with crystal violet clones of tumor cells that colonized the lungs (TCL) were quantified. In these conditions, the number of TCL was reduced by 88% with Zafirlulast compared with 60% with Paclitaxel. The combined treatment of Zafirlukast + Paclitaxel remarkably inhibited by 100% the number of TCL. In contrast, Zafirlukast alone or in combination with Paclitaxel had no significant effect on primary tumor growth. In conclusion we described for the first time that blockade of platelet-CysLT1 receptor altered the protumoral action of platelets and prevented breast cancer cell metastasis to bone and lungs. Citation Format: Lou Saier, Olivier Peyruchaud. Pharmacological blockade of platelet-CysLT1 receptor counteracts platelet protumoral action and prevents breast cancer cell metastasis to bone and lungs [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT011.

Pingyangmycin enhances the antitumor efficacy of anti-PD-1 therapy associated with tumor-infiltrating CD8+ T cell augmentation

To investigate the antitumor efficacy of pingyangmycin (PYM) in combination with anti-PD-1 antibody and determine the capability of PYM to induce immunogenic cell death (ICD) in cancer cells. The murine 4T1 breast cancer and B16 melanoma models were used for evaluation of therapeutic efficacy of the combination of PYM with anti-PD-1 antibody. The ELISA kits were used to quantify the ICD related ATP and HMGB1 levels. The Transwell assay was conducted to determine the chemotaxis ability of THP-1 cell in vitro. The flow cytometry was used to measure reactive oxygen species level and analyze the ratio of immune cell subsets. PYM induced ICD in murine 4T1 breast cancer and B16 melanoma cells and increased the release of nucleic acid fragments that may further promote the monocytic chemotaxis. In the 4T1 murine breast cancer model, PYM alone, anti-PD-1 antibody alone, and their combination suppressed tumor growth by 66.3%, 16.1% and 77.6%, respectively. PYM markedly enhanced the therapeutic efficacy of anti-PD-1 antibody against 4T1 breast cancer. The calculated CDI (coefficient of drug interaction) indicated synergistic effect. Evaluated by graphic analysis, the nucleated cells intensity in the femur bone marrow remained unchanged. Histopathological observations revealed no noticeable toxico-pathological changes in the lung and various organs, indicating that the PYM and anti-PD-1 antibody combination exerted enhanced efficacy at well-tolerated dosage level. By the combination treatment, a panel of immunological changes emerged. The ratio of CD3+ cells, NK cells and NKT cells increased and Tregs decreased in peripheral blood. The DCs increased in the spleen. Prominent changes occurred in tumor infiltrating lymphocytes. The ratio of CD8+ cells increased, while that of CD4+ cells decreased; however, the ratio of CD3+ cells remained unchanged, implying that certain immunological responses emerged in the tumor microenvironment. PYM alone could also increase CD8+ cells and reduce CD4+ cells in tumor infiltrating lymphocytes. The studies indicate that PYM, as an ICD inducer with mild myelosuppression effect, may enhance the therapeutic efficacy of anti-PD-1 antibody in association with tumor infiltrating CD8+ T cell augmentation.

Mitochondrial DNA Affects the Expression of Nuclear Genes Involved in Immune and Stress Responses in a Breast Cancer Model.

Tumor cells without mitochondrial (mt) DNA (ρ0 cells) are auxotrophic for uridine, and their growth is supported by pyruvate. While ATP synthesis in ρ0 cells relies on glycolysis, they fail to form tumors unless they acquire mitochondria from stromal cells. Mitochondrial acquisition restores respiration that is essential for de novo pyrimidine biosynthesis and for mitochondrial ATP production. The physiological processes that underpin intercellular mitochondrial transfer to tumor cells lacking mtDNA and the metabolic remodeling and restored tumorigenic properties of cells that acquire mitochondria are not well understood. Here, we investigated the changes in mitochondrial and nuclear gene expression that accompany mtDNA deletion and acquisition in metastatic murine 4T1 breast cancer cells. Loss of mitochondrial gene expression in 4T1ρ0 cells was restored in cells recovered from subcutaneous tumors that grew from 4T1ρ0 cells following acquisition of mtDNA from host cells. In contrast, the expression of most nuclear genes that encode respiratory complex subunits and mitochondrial ribosomal subunits was not greatly affected by loss of mtDNA, indicating ineffective mitochondria-to-nucleus communication systems for these nuclear genes. Further, analysis of nuclear genes whose expression was compromised in 4T1ρ0 cells showed that immune- and stress-related genes were the most highly differentially expressed, representing over 70% of those with greater than 16-fold higher expression in 4T1 compared with 4T1ρ0 cells. The monocyte recruiting chemokine, Ccl2, and Psmb8, a subunit of the immunoproteasome that generates MHCI-binding peptides, were the most highly differentially expressed. Early monocyte/macrophage recruitment into the tumor mass was compromised in 4T1ρ0 cells but recovered before mtDNA could be detected. Taken together, our results show that mitochondrial acquisition by tumor cells without mtDNA results in bioenergetic remodeling and re-expression of genes involved in immune function and stress adaptation.

Abstract 1476: Vessel normalization following LDH-A knockdown in murine breast tumors

Abstract Abnormal vessel formation plays a major role in tumor progression. Previously, we showed that lactate dehydrogenase A (LDH-A) knockdown (KD) reduces orthotopic 4T1 breast tumor lactate, and has a robust anti-tumor effect through changes in the tumor microenvironment (TME) [1, 2]. Tumors with LDH-A downregulation have lower VEGF-A secretion and reduced formation of new blood vessels [2]. In addition, LDH-A KD cells/tumors displayed reduced HIF-1 signaling, as assessed by BLI using HRE-exGLuc reporter. Changes in the bioluminescence signal corresponded with changes in HIF-1α staining. LDH-A KD tumors also showed less HIF-1α staining compared to control tumors, consistent with less pimonidazole (hypoxia) staining [2]. Murine 4T1 breast cancer cells and tumors with LDH-A knockdown were developed and described [2]. To evaluate the degree of tumor neovascularization and its normalization, we stained tumors for CD31 (endothelial marker) and NG2 (pericytes marker). For pericyte coverage quantification, vessels were counted, and percentage of vessels covered by pericytes were calculated. Blood vessel permeability was assessed by intravenous injection of Evans Blue dye and tumor dye extravasation was quantified. To assess in vivo vascular network over time, a raster-scan optoacoustic mesoscopy (RSOM) was performed. Vessel structure in 4T1 murine tumors changes significantly following LDH-A KD. Normalized LDH-A KD tumor neovascularization was shown using the double immunofluorescence staining for CD31 and NG2, including a more normal vascular morphology, with shorter, more linear (less branched) vessel structure, with a higher percentage of vessels covered by pericytes compared to control tumors. Evans Blue dye extravasation was significantly less (lower vascular permeability) in LDH-A KD tumors compared to control tumors. Applying RSOM to follow the development of the vascular network during tumor growth, we showed better vascularization of LDH-A KD tumors. Furthermore, the number of circulating tumor cells (CTCs) was also significantly lower in blood collected from mice bearing LDH-A KD tumors. We show that LDH-depletion leads to tumor vessel normalization that includes several unique features: structural changes, greater coverage of blood vessel by pericytes and reduced vessel leakiness, leading to changes in the tumor microenvironment, inhibition of metastases formation and prolongation of survival. 1. Rizwan, A., et al., Relationships between LDH-A, lactate, and metastases in 4T1 breast tumors. Clin Cancer Res, 2013. 19(18): p. 5158-69. 2. Serganova, I., et al., LDH-A regulates the tumor microenvironment via HIF-signaling and modulates the immune response. PLOS ONE, 2018. 13(9): p. e0203965. Citation Format: Inna Serganova, Kiranmayi Vemuri, Ivan Cohen, Matthew Lubin, Sheryl Roberts, Masatomo Maeda, Mayuresh Mane, Henry Mann, Thomas Reiner, Eric Chan, Dmitry Yarilin, Katia Manova-Todorova, Roberta Zappasodi, Taha Merghoub, Jason Koutcher, Ronald Blasberg. Vessel normalization following LDH-A knockdown in murine breast tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1476.

Endogenous PAD4 in Breast Cancer Cells Mediates Cancer Extracellular Chromatin Network Formation and Promotes Lung Metastasis.

Peptidyl arginine deiminase 4 (PAD4/PADI4) is a posttranslational modification enzyme that converts protein arginine or mono-methylarginine to citrulline. The PAD4-mediated hypercitrullination reaction in neutrophils causes the release of nuclear chromatin to form a chromatin network termed neutrophil extracellular traps (NET). NETs were first described as antimicrobial fibers that bind and kill bacteria. However, it is not known whether PAD4 can mediate the release of chromatin DNA into the extracellular space of cancer cells. Here, we report that murine breast cancer 4T1 cells expressing high levels of PADI4 can release cancer extracellular chromatin networks (CECN) in vitro and in vivo. Deletion of Padi4 using CRISPR/Cas9 abolished CECN formation in 4T1 cells. Padi4 deletion from 4T1 cells also reduced the rate of tumor growth in an allograft model, and decreased lung metastasis by 4T1 breast cancers. DNase I treatment, which degrades extracellular DNA including CECNs, also reduced breast to lung metastasis of Padi4 wild-type 4T1 cells in allograft experiments in the Padi4-knockout mice. We further demonstrated that DNase I treatment in this mouse model did not alter circulating tumor cells but decreased metastasis through steps after intravasation. Taken together, our genetic studies show that PAD4 plays a cell autonomous role in cancer metastasis, thus revealing a novel strategy for preventing cancer metastasis by inhibiting cancer cell endogenous PAD4. IMPLICATIONS: This study shows that PADI4 can mediate the formation of CECNs in 4T1 cells, and that endogenous PADI4 plays an essential role in breast cancer lung metastasis. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/5/735/F1.large.jpg.

Targeting GARP-TGFβ axis enhances anti-tumor efficacy of immune checkpoint blockade therapy

Abstract Although immune checkpoint blockade (ICB) has emerged as a promising cancer therapy, a majority of cancers, such as pancreatic cancer and triple negative breast cancer, fail to respond to ICB. A potential mechanism is the accumulation of transforming growth factor-β1 (TGFβ1) in tumor microenvironment (TME) which drives immune dysfunction by inducing regulatory T cells (Treg), excluding and inhibiting the function of effector CD8+ T cells. Glycoprotein-A repetitions predominant protein (GARP) is a cell surface docking and activating receptor for latent TGFβ1 and highly expressed in breast cancer cells, Tregs and platelets. Previous work from our lab has demonstrated a critical role for GARP in dampening anti-tumor immunity via TGFβ1 pathway. To validate if GARP is a therapeutic target, we generated an anti-human GARP antibody (4D3). We found that 4D3 blocked GARP-latent TGFβ1 interaction and limited active TGFβ1 release. We expressed human GARP in murine 4T1 breast cancer cells. In this orthotopic syngeneic mouse tumor model, combination therapy with 4D3 and anti-PD1 antibody suppressed tumor growth and metastasis more effectively than treatment with either agent alone, resulting in significantly enhanced survival. Mechanistically, the combination therapy reduced p-SMAD3 expression as well as collagen deposition in the TME. In addition, 4D3 treatment reduced tumor-associated macrophages and their PD-L1 expression. Collectively, our preclinical data demonstrated that simultaneous blockade of the PD1 and GARP-TGFβ1 axis elicits potent and superior anti-tumor efficacy relative to monotherapies, suggesting a potentially more effective strategy against cancers that are resistant to current immune checkpoint blockade.