Syngeneic Mouse Model Of Breast Cancer Research Articles

Taxane chemotherapy induces stromal injury that leads to breast cancer dormancy escape.

A major cause of cancer recurrence following chemotherapy is cancer dormancy escape. Taxane-based chemotherapy is standard of care in breast cancer treatment aimed at killing proliferating cancer cells. Here, we demonstrate that docetaxel injures stromal cells, which release protumor cytokines, IL-6 and granulocyte colony stimulating factor (G-CSF), that in turn invoke dormant cancer outgrowth both in vitro and in vivo. Single-cell transcriptomics shows a reprogramming of awakened cancer cells including several survival cues such as stemness, chemoresistance in a tumor stromal organoid (TSO) model, as well as an altered tumor microenvironment (TME) with augmented protumor immune signaling in a syngeneic mouse breast cancer model. IL-6 plays a role in cancer cell proliferation, whereas G-CSF mediates tumor immunosuppression. Pathways and differential expression analyses confirmed MEK as the key regulatory molecule in cancer cell outgrowth and survival. Antibody targeting of protumor cytokines (IL-6, G-CSF) or inhibition of cytokine signaling via MEK/ERK pathway using selumetinib prior to docetaxel treatment prevented cancer dormancy outgrowth suggesting a novel therapeutic strategy to prevent cancer recurrence.

Taxifolin Inhibits Breast Cancer Growth by Facilitating CD8+ T Cell Infiltration and Inducing a Novel Set of Genes including Potential Tumor Suppressor Genes in 1q21.3.

Taxifolin inhibits breast cancer (BC) via novel mechanisms. In a syngeneic mouse BC model, taxifolin suppressed 4T-1 cell-derived allografts. RNA-seq of 4T-1 tumors identified 36 differentially expressed genes (DEGs) upregulated by taxifolin. Among their human homologues, 19, 7, and 2 genes were downregulated in BCs, high-proliferative BCs, and BCs with high-fatality risks, respectively. Three genes were established as tumor suppressors and eight were novel to BC, including HNRN, KPRP, CRCT1, and FLG2. These four genes exhibit tumor suppressive actions and reside in 1q21.3, a locus amplified in 70% recurrent BCs, revealing a unique vulnerability of primary and recurrent BCs with 1q21.3 amplification with respect to taxifolin. Furthermore, the 36 DEGs formed a multiple gene panel (DEG36) that effectively stratified the fatality risk in luminal, HER2+, and triple-negative (TN) equivalent BCs in two large cohorts: the METABRIC and TCGA datasets. 4T-1 cells model human TNBC cells. The DEG36 most robustly predicted the poor prognosis of TNBCs and associated it with the infiltration of CD8+ T, NK, macrophages, and Th2 cells. Of note, taxifolin increased the CD8+ T cell content in 4T-1 tumors. The DEG36 is a novel and effective prognostic biomarker of BCs, particularly TNBCs, and can be used to assess the BC-associated immunosuppressive microenvironment.

Pharmacological anti-tumor effects of natural Chamaecyparis obtusa (siebold & zucc.) endl. Leaf extracts on breast cancer

Ethnopharmacological relevanceChamaecyparis obtusa (C. obtusa, cypress species) is a plant that grows mainly in the temperate Northern Hemisphere and has long been used as a traditional anti-inflammatory treatment in East Asia. C. obtusa contains phytoncides, flavonoids, and terpenes, which have excellent anti-cancer effects and have been reported to prevent the progression of various cancers. However, the detailed mechanisms underlying the anti-cancer effects of C. obtusa extracts are unknown. Aim of the studyWe sought to confirm the anti-cancer effects of C. obtusa leaf extracts and to reveal the mechanism of action, with the possibility of its application in the treatment or prevention of cancer. Material &methodsThe cytotoxicity of C. obtusa leaf extracts was confirmed using an MTT assay. Intracellular changes in protein levels were measured by immunoblotting, and mRNA levels were measured with qRT-PCR. Wound healing assay and transwell migration assay were used to evaluate the metastatic potential of breast cancer cells. The extract-induced apoptosis was observed using IncuCyte Annexin V Red staining analysis. A syngeneic breast cancer mouse model was established by injecting 4T1-Luc mouse breast cancer cells into the fat pad of female BALB/c mice, and the extract was administered orally. Luciferin solution was injected intraperitoneally to assess primary tumor development and metastasis by bioluminescence. ResultsC. obtusa leaf extracts were extracted with boiling water, 70% EtOH, and 99% EtOH. Among the extracts, the 99% EtOH extract of C. obtusa leaf (CO99EL) most clearly inhibited the tyrosine phosphorylation of Signal Transducer and Activator of Transcription 3 (pY-STAT3) in MDA-MB-231 breast cancer cells at a concentration of 25 and 50 μg/mL. In addition, CO99EL strongly inhibited not only endogenous pY-STAT3 levels but also IL-6-induced STAT3 activation in various types of cancer cells, including breast cancer. CO99EL inhibited metastatic potential by downregulating the expression of N-cadherin, fibronectin, TWIST, MMP2, and MMP9 in MDA-MB-231 breast cancer cells. CO99EL also induced apoptotic cell death by increasing cleaved caspase-3 and decreasing anti-apoptotic proteins Bcl-2 and Bcl-xL. In an in vivo syngeneic breast cancer mouse model, 100 mg/kg CO99EL suppressed tumor growth and induced apoptosis of cancer cells. Moreover, CO99EL significantly inhibited lung metastasis from primary breast cancer. ConclusionsOur study demonstrated that 100 mg/kg CO99EL has potent anti-tumor effects against breast cancer, thus suggesting that 100 mg/kg CO99EL has potential applications in the treatment and prevention of breast cancer.

Abstract P2-20-05: Personalized tumor vaccine can suppress tumor growth and metastasis in the syngeneic mouse breast cancer model

Abstract Objective Immunotherapy using the tumor-specific antigens (TSAs) is a promising strategy in breast cancer. Studies have suggested that the in vivo exposures to certain tumors can induce adaptive anti-tumor immunity in syngeneic tumor models. In this study, we show the efficacy of the tumor lysate vaccine and peptide-based vaccine against tumor neoantigen in suppressing tumor growth and metastasis in 4T1 syngeneic tumor models. Method We used BALB/c mice and its syngeneic tumor cell lines to evaluate the anti-tumor effect induced by the transient exposure to the tumor cells. For tumor vaccines, we synthesized the tumor lysate vaccine by the freeze-thaw method or synthetic peptide against the selected tumor neoantigens identified by exome sequencing. We systemic and local immune remodeling was investigated by using immunohistochemistry, flow cytometry, and single cell RNA sequencing. Results We observed a significant reduction of tumor growth and metastasis in 4T1 syngeneic tumors when the mice were previously exposed to the same cells (pre-exposure group). This anti-tumor effect induced by the exposures to the tumor was cell line-specific. The 4T1 tumor lysate vaccines administered prior to the tumor cell injection also showed significant inhibitory effect on tumor growth and metastasis. T lymphocytes, isolated from the tumor tissues of the 4T1 pre-exposure mice and lysate vaccine-treated mice, showed higher levels of TNF-ɑ and IFN-ɣ when compared to the control those from the control tumors. The lysate vaccine treatment resulted in a substantial remodeling of tumor microenvironment including reduction of myeloid-derived suppressor cells and M2 tumor-associated macrophages. On the other hand, the numbers of M1 tumor-associated macrophages and effector memory CD8+ T cells were increased by the lysate vaccine. While the peptide vaccine showed no inhibitory effect on the primary tumor growth, it also suppressed spontaneous lung metastasis. Finally, we administered lysate tumor vaccine after the tumor establishment to determine the therapeutic effect. The lysate vaccine significantly suppressed the tumor growth and lung metastasis of the syngeneic 4T1 tumors. Conclusion Tumor lysate vaccine can suppress the tumor growth and metastasis in the 4T1 syngeneic mouse models by inducing substantial remodeling of tumor immune microenvironment. Additionally, tumor lysate vaccine can elicit similar anti-tumor immune response when administered after the establishment of the primary tumor suggesting a potential therapeutic value. Citation Format: Hyeong-Gon Moon, Hye Youn Son, Woo Hang Heo, Mingji Quan, SONGBIN LI, Haritonova Valentina, Hamin Jeong, Wonshik Han, Han-Byoel Lee, YUJEONG HER, Ju Hee Kim. Personalized tumor vaccine can suppress tumor growth and metastasis in the syngeneic mouse breast cancer model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-05.

A new miRNA-Modified coxsackievirus B3 inhibits triple negative breast cancer growth with improved safety profile in immunocompetent mice

Coxsackievirus B3 (CVB3) displays great oncolytic activity against various cancer cells. Previously, we demonstrated that adding targeting sequences (TS) of miR-145/143, which are downregulated in cancer compared with normal cells, into CVB3 genome drastically attenuates tissue toxicity, while retaining its oncolytic activity towards lung tumor. Here we extended to assess miR-modified CVB3 in breast cancer therapy. We generated a new miRNA-CVB3 by inserting TS of muscle-specific miR-1 and pancreas-selective miR-216 into the above miR-145/143-modified CVB3. We found that this newly established CVB3 (termed miR-CVB3-1.1) is safe without triggering noticeable pathogenesis when applied to immunocompetent mice. In vitro studies revealed that miR-CVB3-1.1 can infect and lyse a wide range of breast cancer cells. Animal experiments using a syngeneic breast cancer mouse model showed that intratumoral inoculation of miR-CVB3-1.1 significantly suppresses tumor growth and metastasis, associated with productive viral growth and enhanced immune cell infiltration in the tumor microenvironment. Moreover, we observed substantially reduced toxicity and prolonged survival in mice treated with miR-CVB3-1.1 compared with wild-type CVB3. Together, our results support miR-CVB3-1.1 as a promising candidate, which can be further evaluated for clinical treatment of breast cancer.

Abstract 3454: Adenosine receptor antagonists A2AR (TT-10) and A2BR (TT-4) demonstrate anti-tumor activity in 4T1-induced syngeneic breast cancer mouse model

Abstract Background: Extracellular adenosine has been shown to negatively regulate anti-tumor immunity. Adenosine is produced at high levels in the TME as a result of hypoxia-driven tumor cell death. As a result, ATP is released from dying cells generating adenosine via enzymatic dephosphorylation, mainly by ectonucleotidases CD39 & CD73. This results in high extracellular adenosine levels, being elevated 100-500x in the TME. The free adenosine proceeds to binding one of its four known receptors A1, A2a, A2b & A3. The binding of adenosine to A2AR & A2BR contributes to immunosuppression via cyclic AMP pathway. It has been observed that bound A2AR suppress T cell function (CD8+T & NK) while A2BR dampens myeloid cell function and promotes chemotaxis. These concerted actions thereby enhance tumor growth and metastasis. Blocking the binding of adenosine to the A2AR & A2BR results in T-cell activation and infiltration of immune cells in the TME. TT-10 & TT-4 are potent and selective antagonists of A2AR & A2BR. Both agents are being developed for the treatment of advanced cancers individually and as dual inhibitors. Methods: Balb/c mice were implanted with 5 x 104 4T1 cells and randomly assigned to 6 groups per study; (1) Vehicle Control-VC, (2) TT-10, 1 mg/kg, PO, BID (3) TT-4, 3 mg/kg, PO, BID (4) Anti-mPD-1, 200 ug, IP (D4, 8, 14) (5) 1 mg/kg TT-10 + 200 ug Anti-mPD-1 (D4, 8, 14) and (6) TT-4, 3 mg/kg, PO, BID + 200 ug Anti-mPD-1 (D4, 8, 14). Mice were dosed for 21 days, and tumor volume was measured 2x a week. After 21 days post initiation of treatment (31 days post 4T1 cell inoculation), mice were sacrificed, TIL populations were isolated from 4T1 induced tumors and interrogated via Flow. Markers for surface staining included CD45, CD3, CD4, CD8, CD11b, CD49, Ly6G. Results: All implanted mice developed measurable tumors. Mean suppression of tumor growth was observed to be greater in single agent TT-10 (p<0.05) & TT-4 (p<0.05), over single agent anti-PD-1 inhibitor when compared to the control on day 31, post 4T1 cell inoculation. Analysis of the TIL at the end of the study further supported the observation of tumor growth suppression, from increased T-cell activity seen for both TT-10 & TT-4 when compared to the VC. Furthermore, striking reductions in MDSC populations were also observed in both TT-10 & TT-4 treated mice, when compared to single agent anti-PD-1 and the VC. Conclusions: TT-10 & TT-4 alone was superior (p<0.05) to the VC as well as single agent anti-PD-1 in slowing tumor growth, which was further supported by the increase in T-cell activity observed in the end of study TIL analysis. Lastly, striking reductions in MDSC populations were also observed in both TT-10 & TT-4 treated mice, when compared to single agent anti-PD-1 and the VC. Citation Format: Desa Rae E. Pastore, Kasim Mookhtiar, Brian Schwartz, Sushant Kumar, Ranganayaki Nagaraj, Ashwinkumar V. Meru. Adenosine receptor antagonists A2AR (TT-10) and A2BR (TT-4) demonstrate anti-tumor activity in 4T1-induced syngeneic breast cancer mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3454.

Abstract P1-06-02: Targeted deletion of Kindlin-2 in mouse mammary glands inhibits tumor growth, invasion and metastasis downstream of TGF-β/EGF oncogenic signaling pathway

Abstract Cancer metastasis is a complex process by which cancer cells migrate through the blood and the lymphatic systems to lodge and proliferate in distant sites and organs in the body. Metastasis is the main cause of death in patients suffering from cancer, including those patients with breast cancer. Breast cancer (BC) is the most frequently diagnosed malignancy in women and is one of the leading causes of death due to cancer invasion, metastasis, and resistance to therapies. Among its variants, triple-negative breast cancer (TNBC) is considered the most aggressive due to its early invasive and metastatic properties with poor prognosis. Kindlin-2, which is encoded by Fermitin family homolog 2 gene (FERMT2) has been associated with pathogenesis of several types of cancers of epithelial origin. Our previous studies have addressed the role of Kindlin-2 as a major regulator of the invasion-metastasis cascade in breast cancer by controlling several hallmarks of cancer in tumor cells. The contribution of mammary epithelial cell Kindlin-2 in the mammary glands to the process of tumor progression and metastasis has, however, not been investigated. Accordingly, we generated a floxed mouse strain by targeting the FREMT2 (K2lox/lox) locus using a CRISPR/Cas9-based editing strategy, followed by tissue-specific deletion of the Kindlin-2 in the basal subtype of the mammary epithelial cells (MECs) in the mouse mammary glands by crossing the K2lox/lox mice with K14-cre mice. Loss of Kindlin-2 in the basal MECs had no deleterious effects on mammary glands development, mouse development and fertility and lactation in mice bearing the Kindlin 2-deletetion phenotype and their progeny. However, in a syngeneic mouse model of BC, the loss of Kindlin-2 in MECs inhibited tumor growth and metastasis in mice inoculated with the aggressive murine TNBC E0771 cells when implanted directly in their mammary fat pads. Injecting the E0771 cells via the tail vein of Kindlin-2-deleted mice had, however, no effect on tumor colonization in the lungs, when compared to wild-type mice, clearly supporting a critical role of MECs Kindlin-2 in BC tumor growth and metastasis. Mechanistically, we found the MECs Kindlin-2-mediated inhibition of tumor growth and metastasis is through the regulation of the TGF-β/ERK MAP. kinase signaling axis, in a similar manner that our published studies showed that Kindlin-2 regulates this oncogenic pathway in the BC cells. Thus, our findings strongly suggest that Kindlin-2 supports BC oncogenesis in both the tumor cells and the MECs in the mammary glands, through the regulation of the TGF-β/EGF oncogenic signaling pathway. Therefore, therapeutic strategies targeting Kindlin-2 in both the cancer cells and the mammary glands may be necessary for a successful inhibition of BC tumors. Citation Format: Priyanka Rana, Wei Wang, Akram Alkrekchi, Katarzyna Bialkowska, Vesna Markovic, Edward F Plow, Elzbieta Pluskota, Khalid Sossey-Alaoui. Targeted deletion of Kindlin-2 in mouse mammary glands inhibits tumor growth, invasion and metastasis downstream of TGF-β/EGF oncogenic signaling pathway [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-06-02.

Targeted Deletion of Kindlin-2 in Mouse Mammary Glands Inhibits Tumor Growth, Invasion, and Metastasis Downstream of a TGF-β/EGF Oncogenic Signaling Pathway.

Simple SummaryStudies from our group and others have established Kindlin-2 that is expressed in the cancer cells as a major driver of tumor progression and metastasis in breast cancer (BC). The role of Kindlin-2 that is expressed in the mammary glands (tumor microenvironment) in the pathogenesis of BC has, however, not been investigated. To this end, we generated a mouse strain that specifically lacks expression of Kindlin-2 in the basal cells within the mouse mammary glands. Loss of Kindlin-2 in this mammary gland compartment, while having no effect on mouse development, significantly inhibited BC tumors growth and metastasis when these Kindlin-2-deficient mice were challenged with mammary fat pad injection of cancer cells. At the molecular signaling level, we found that Kindlin-2 plays a significant role in regulating BC progression and metastasis in both the cancer cells and the tumor microenvironment (mammary epithelial cells) downstream of a TGF-β/EGF signaling axis.Breast cancer (BC) is one of the leading causes of cancer-related deaths due in part to its invasive and metastatic properties. Kindlin-2 (FERMT2) is associated with the pathogenesis of several cancers. Although the role of Kindlin-2 in regulating the invasion-metastasis cascade in BC is widely documented, its function in BC initiation and progression remains to be fully elucidated. Accordingly, we generated a floxed mouse strain by targeting the Fermt2 (K2lox/lox) locus, followed by tissue-specific deletion of Kindlin-2 in the myoepithelial compartment of the mammary glands by crossing the K2lox/lox mice with K14-Cre mice. Loss of Kindlin-2 in mammary epithelial cells (MECs) showed no deleterious effects on mammary gland development, fertility, and lactation in mice bearing Kindlin-2-deletion. However, in a syngeneic mouse model of BC, mammary gland, specific knockout of Kindlin-2 inhibited the growth and metastasis of murine E0771 BC cells inoculated into the mammary fat pads. However, injecting the E0771 cells into the lateral tail vein of Kindlin-2-deleted mice had no effect on tumor colonization in the lungs, thereby establishing a critical role of MEC Kindlin-2 in supporting BC tumor growth and metastasis. Mechanistically, we found the MEC Kindlin-2-mediated inhibition of tumor growth and metastasis is accomplished through its regulation of the TGF-β/ERK MAP kinase signaling axis. Thus, Kindlin-2 within the mammary gland microenvironment facilitates the progression and metastasis of BC.

PKC agonism restricts innate immune suppression, promotes antigen cross-presentation and synergizes with agonistic CD40 antibody therapy to activate CD8+ T cells in breast cancer

Myeloid-derived suppressor cells (MDSCs) are an immature innate cell population that expands in pathological conditions such as cancer and suppresses T cells via production of immunosuppressive factors. Conversely, efficient cytotoxic T cell priming is dependent on the ability of antigen-presenting cells (APCs) to cross-present tumor antigens to CD8+ T cells, a process that requires a specific subtype of dendritic cells (DCs) called conventional DC1 (cDC1) which are often dysfunctional in cancer. One way to activate cDC1 is ligation of CD40 which is abundantly expressed by myeloid cells and its agonism leads to myeloid cell activation. Thus, targeting MDSCs while simultaneously expanding cross-presenting DCs represents a promising strategy that, when combined with agonistic CD40, may result in long-lasting protective immunity. In this study, we investigated the effect of PKC agonists PEP005 and prostratin on MDSC expansion, differentiation, and recruitment to the tumor microenvironment. Our findings demonstrate that PKC agonists decreased MDSC expansion from hematopoietic progenitors and induced M-MDSC differentiation to an APC-like phenotype that expresses cDC1-related markers via activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Simultaneously, PKC agonists favored cDC1 expansion at the expense of cDC2 and plasmacytoid DCs (pDC). Functionally, PKC agonists blunted MDSC suppressive activity and enhanced MDSC cross-priming capacity both in vitro and in vivo. Finally, combination of PKC agonism with agonistic CD40 mAb resulted in a marked reduction in tumor growth with a significant increase in intratumoral activated CD8+ T cells and tissue-resident memory CD8+ T cells in a syngeneic breast cancer mouse model. In sum, this work proposes a novel promising strategy to simultaneously target MDSCs and promote APC function that may have highly impactful clinical relevance in cancer patients.

Transfection with p21 and p53 tumor suppressor plasmids suppressed breast tumor growth in syngeneic mouse model

Treatment of breast cancer by delivering important tumor suppressor plasmids is a promising approach in the field of clinical medicine. We transfected p21 and p53 tumor suppressor plasmids, into different breast cancer cell lines using inorganic nanoparticles (NPs) of carbonate apatite to evaluate the effect of gene expression on reducing breast cancer cell growth. In triple negative MDA-MB-231 breast cancer cell line, the cytotoxicity assay upon combined delivery of p21 and p53 plasmid loaded NPs showed significant decrease in cell growth compared to distinct p21 or p53 treatments. Also, in MCF-7 and 4T1 cell lines, significant reduction in cellular growth was observed following p21 or p53 plasmid transfection. The Western blot data showed that NP loaded p21 and p53 transgene delivery in MDA-MB-231 cell line resulted in a noteworthy decrease in phosphorylated form of MAPK protein of MAPK/ERK pathway. The in vivo studies in syngeneic breast cancer mouse model demonstrated that the rate of growth and final tumor volume were reduced to a greater extent in mice that received intravenous injection of p21 + NP and p53 + NP therapeutics.

SiRNAs targeting multidrug transporter genes sensitise breast tumour to doxorubicin in a syngeneic mouse model

Chemotherapy, the commonly favoured approach to treat cancer is frequently associated with treatment failure and recurrence of disease as a result of development of multidrug resistance (MDR) with concomitant over-expression of drug efflux proteins on cancer cells. One of the most widely used drugs, doxorubicin (Dox) is a substrate of three different ATP-binding cassette (ABC) transporters, namely, ABCB1, ABCG2 and ABCC1, predominantly contributing to MDR phenotype in cancer. To silence these transporter-coding genes and thus enhance the therapeutic efficacy of Dox, pH-sensitive carbonate apatite (CA) nanoparticles (NPs) were employed as a carrier system to co-deliver siRNAs against these genes and Dox in breast cancer cells and in a syngeneic breast cancer mouse model. siRNAs and Dox were complexed with NPs by incubation at 37 °C and used to treat cancer cell lines to check cell viability and caspase-mediated signal. 4T1 cells-induced breast cancer mouse model was used for treatment with the complex to confirm their action in tumour regression. Smaller (∼200 nm) and less polydisperse NPs that were taken up more effectively by tumour tissue could enhance Dox chemosensitivity, significantly reducing the tumour size in a very low dose of Dox (0.34 mg/kg), in contrast to the limited effect observed in breast cancer cell lines. The study thus proposes that simultaneous delivery of siRNAs against transporter genes and Dox with the help of CA NPs could be a potential therapeutic intervention in effectively treating MDR breast cancer.

Abstract 69: Chemotherapy-induced cancer cell death diminishes therapeutic efficacy through heme oxygenase-1-mediated inactivation of M1-like tumor associated macrophages

Abstract One of the most common treatment options for cancer is a chemotherapy. After chemotherapy, however, unwanted host effects provoke tumor recurrence and aggressiveness of cancer cells, which often arise as a consequence of disruption in the patient's immune system. Lymphocytes, such as CD8+ cytotoxic T lymphocytes (CTLs), which have capability of suppressing cancer progression, are depleted following chemotherapy. Conventional chemotherapeutic agents kill cancer cells, which boosts recruitment of myeloid cells. Tumor-associated macrophages (TAMs), emerging as abundant tumor-infiltrating myeloid cells in tumor microenvironment, play an important role in immunosuppression caused by conventional chemotherapy. Chemotherapy-induced dying cancer cells could hijack accumulated TAMs, provoking tumor recurrence. Therefore, reprogramming of TAMs to maximize the chemotherapeutic efficacy is considered a promising novel anticancer strategy. In this study, we investigated whether dying cancer cells after chemotherapy could reduce therapeutic efficacy by modulating tumor-infiltrating macrophage activity. In a 4T1 syngeneic mouse breast cancer model, there was a marked decrease in the proportion of M1-like TAMs expressing CD86 with tumor-suppressive activity and reduced tumor infiltration of CTLs following treatment with paclitaxel. Paclitaxel treatment also resulted in an enhancement of heme oxygenase-1 (HO-1) mRNA levels in tumor-infiltrating myeloid cells engulfing dying cancer cells. Consistent with the in vivo profile of TAMs, bone marrow-derived macrophages (BMDMs) phagocytosing dying breast cancer cells exhibited significant upregulation of HO-1 expression. HO-1 induction in BMDMs engulfing dying breast cancer cells facilitated M2 polarization. In contrast, abrogation of HO-1 activity sustained M1 activation of BMDMs co-cultured with dying breast cancer cells. Pharmacologic inhibition of HO-1 activity with zinc protoporphyin IX augmented paclitaxel efficacy by stimulating tumor infiltration of CTLs in a 4T1 breast cancer model. Furthermore, blockade of HO-1 in breast tumor-bearing mice induced increased M1-like TAMs expressing CD86 that plays an important role in stimulating antitumor immune response. Taken together, HO-1 induction by the chemotherapy-derived dying cancer cells in TAMs dampens antitumor effects of chemotherapy by manipulating innate and adaptive immunity. Our results demonstrate that targeting overexpressed HO-1 in the breast tumor microenvironment can control the immune system to potentiate the efficacy of chemotherapy. Citation Format: Seung Hyeon Kim, Xiancai Zhong, Shin-Young Gwak, Ishrat Aklima Muna, Sin-Aye Park, Young-Nam Cha, Young-Joon Surh. Chemotherapy-induced cancer cell death diminishes therapeutic efficacy through heme oxygenase-1-mediated inactivation of M1-like tumor associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 69.

A Novel Benzocoumarin-Stilbene Hybrid as a DNA ligase I inhibitor with in vitro and in vivo anti-tumor activity in breast cancer models

Existing cancer therapies are often associated with drug resistance and toxicity, which results in poor prognosis and recurrence of cancer. This necessitates the identification and development of novel therapeutics against existing as well as novel cellular targets. In this study, a novel class of Benzocoumarin-Stilbene hybrid molecules were synthesized and evaluated for their antiproliferative activity against various cancer cell lines followed by in vivo antitumor activity in a mouse model of cancer. The most promising molecule among the series, i.e. compound (E)-4-(3,5-dimethoxystyryl)-2H-benzo[h]chromen-2-one (19) showed maximum antiproliferative activity in breast cancer cell lines (MDA-MB-231 and 4T1) and decreased the tumor size in the in-vivo 4T1 cell-induced orthotopic syngeneic mouse breast cancer model. The mechanistic studies of compound 19 by various biochemical, cell biology and biophysical approaches suggest that the compound binds to and inhibits the human DNA ligase I enzyme activity that might be the cause for significant reduction in tumor growth and may constitute a promising next-generation therapy against breast cancers.

Abstract 4776: Circulating clusters in breast cancer express cancer stem cell phenotypes

Abstract Breast cancer is a leading cause of death in women and its metastatic spread to distant organs is responsible for over 90% of these deaths. Circulating tumor cells (CTCs) implicated in the metastatic process occur when cancer cells undergo cytoskeletal and phenotypic transitions that enhance motility, then penetrate into the blood circulation. While CTC dissemination has typically been considered as the spread of single cells, recent evidence suggests that collective migration results in polyclonal circulating clusters that are the main drivers of metastasis. To study circulating clusters we have developed a dual microfiltration system employing two stages of antigen-independent mechanical cell isolation. As whole blood is passed through the system, circulating clusters are trapped by the first stage while single CTCs are retained by the second stage. Characterization experiments using cell lines demonstrated a capture efficiency of 87.1% for clusters and 85.7% for single cells on their respective microfilters. This system was used to investigate circulating cells in the metastatic MMTV-PyMT syngeneic mouse breast cancer model. Both clusters and single CTCs were detected in mice with histological evidence of lung metastases (14-16 weeks old), but not in mice that had not yet developed metastatic tumors (<12 weeks old). Immunofluorescence assays were established to determine expression of CD24, CD90, cytokeratins, CD45 and DAPI. Tumor cells were identified as nucleated cells that expressed epithelial cytokeratins, but not the leukocyte antigen CD45. While PyMT cells with a CD90+/CD24+ stem cell phenotype comprise only ~1.5% of tumors, they were observed in > 50% of circulating clusters. The presence of cancer stem cells was correlated to cluster size, with smaller clusters containing a greater percentage of CD90+/CD24+ cells. Isolated clusters will be further analyzed to quantify expression levels of cancer stem cell markers in each individual constituent cell. The analysis of circulating clusters may provide crucial insight into the mechanisms of metastatic disease and provide the means to discover novel targets for therapeutic intervention. Citation Format: Ramdane Harouaka, Gabrielle Hodges, Allison David, Rishika Polasani, Spencer Freeman, Katherine Sanchez, Kaitlin Harvey, Max S. Wicha. Circulating clusters in breast cancer express cancer stem cell phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4776. doi:10.1158/1538-7445.AM2017-4776

Chemical Approach to Positional Isomers of Glucose-Platinum Conjugates Reveals Specific Cancer Targeting through Glucose-Transporter-Mediated Uptake in Vitro and in Vivo.

Glycoconjugation is a promising strategy for specific targeting of cancer. In this study, we investigated the effect of d-glucose substitution position on the biological activity of glucose-platinum conjugates (Glc-Pts). We synthesized and characterized all possible positional isomers (C1α, C1β, C2, C3, C4, and C6) of a Glc-Pt. The synthetic routes presented here could, in principle, be extended to prepare glucose conjugates with different active ingredients, other than platinum. The biological activities of the compounds were evaluated both in vitro and in vivo. We discovered that varying the position of substitution of d-glucose alters not only the cellular uptake and cytotoxicity profile but also the GLUT1 specificity of resulting glycoconjugates, where GLUT1 is glucose transporter 1. The C1α- and C2-substituted Glc-Pts (1α and 2) accumulate in cancer cells most efficiently compared to the others, whereas the C3-Glc-Pt (3) is taken up least efficiently. Compounds 1α and 2 are more potent compared to 3 in DU145 cells. The α- and β-anomers of the C1-Glc-Pt also differ significantly in their cellular uptake and activity profiles. No significant differences in uptake of the Glc-Pts were observed in non-cancerous RWPE2 cells. The GLUT1 specificity of the Glc-Pts was evaluated by determining the cellular uptake in the absence and in the presence of the GLUT1 inhibitor cytochalasin B, and by comparing their anticancer activity in DU145 cells and a GLUT1 knockdown cell line. The results reveal that C2-substituted Glc-Pt 2 has the highest GLUT1-specific internalization, which also reflects the best cancer-targeting ability. In a syngeneic breast cancer mouse model overexpressing GLUT1, compound 2 showed antitumor efficacy and selective uptake in tumors with no observable toxicity. This study thus reveals the synthesis of all positional isomers of d-glucose substitution for platinum warheads with detailed glycotargeting characterization in cancer.

Abstract P6-13-17: The combination of eribulin and everolimus results in enhanced suppression of tumors in mouse models of triple negative breast cancer

Abstract INTRODUCTION. Triple negative breast cancer (TNBC) is an aggressive form of breast cancer with poor overall and relapse free survival. TNBC does not have targeted or matched therapies. Patients have worse outcomes after chemotherapy than with other subtypes of breast cancer. TNBC accounts for 12-17% of all breast cancers, leaving an unmet need for targeted therapy. Efforts to profile these tumors have revealed several potential targets. The PI3K/AKT/mTOR pathway is a signal transduction pathway that links growth related hormone receptor interaction to downstream targets such as AKT and mammalian target of rapamycin (mTOR). This pathway targets affect cell proliferation, survival, and apoptosis. Patients with TNBC have high levels of AKT expression and activation of this pathway. Microtubule-targeting agents have been used in TNBC. Eribulin mesylate is a microtubule-targeting agent with benefits in treating taxane and anthracycline refractory breast cancer via a microtubule targeting anti-mitotic mechanism. It has been approved for the treatment of TNBC in heavily pretreated patients. Despite targeted therapy, breast cancer cells can grow resistant. Targeting multiple cancer growth pathways has been used in patients that progress on therapy or fail to respond. We hypothesized that targeting both mitotic blockade and PI3K/AKT/mTOR pathway may provide enhanced suppression of TNBC growth in both syngeneic and xenogeneic mouse models. MATERIALS AND METHODS. MDA-MB-468 is a human TNBC cell line. 4T1 is a highly metastatic mouse TNBC cell line derived from a spontaneously arising Balb/c mammary tumor. 4T1 and MDA-MB-468 tumor cells were injected into the mammary fat pad of female Balb/c and NOD/SCID/IL2Rgamma null (NSG) mice (with matrigel) respectively. After tumors were formed Balb/c mice were treated three times per week with vehicle, eribulin (0.75 mg/kg i.v.), RAD001 (5 mg/kg via oral gavage) or a combination of both. NSG mice were treated three times per week with vehicle, eribulin (0.5 mg/kg i.v.), RAD001 (5 mg/kg by oral gavage), or a combination of both. Tumor volumes and body weights were measured. Student t-test was used to compare the means of two groups and determine the p value (p<0.05 is significant). N=3-8 per group. Table I. 4T1 mouse breast cancer modelTreatmentTumor Volume (mm3)+/-SEMVehicle511.6+/-56.82Eribulin445.6+/-92.17Everolimus324.9+/-24.55Combination171.4+/-16.07 p valueCombination vs. Vehicle0.0001Combination vs. Eribulin0.01Combination vs. Everolimus0.001 Table II. MDA-MB-468 human breast cancer cells in immune deficient mice.TreatmentTumor Volume (mm3)+/-SEMVehicle966.8+/-69.2Eribulin67.81+/-11.79Everolimus830.6+/-156.3Combination31.37+/-3.37 p valueCombination vs. Eribulin0.041Combination vs. Everolimus0.0076 RESULTS. In the 4T1 syngeneic breast cancer mouse model, the combination of Eribulin and Everolimus resulted in marked suppression of tumor growth which was statistically significant versus vehicle treatment alone, or Eribulin or Everolimus alone (Table I). In the MDA-MB-468 model, the combination of Eribulin and Everolimus demonstrated marked suppression of tumor growth which was statistically significant compared to either agent alone (Table II). Citation Format: Marcinkowski E, Luu T, Yuan Y, Mortimer J, Leong L, Portnow J, Xing Q, Wen W, Yim J. The combination of eribulin and everolimus results in enhanced suppression of tumors in mouse models of triple negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-13-17.

Abstract PR01: Rebastinib, a selective TIE2 kinase inhibitor, decreases TIE2-expressing macrophages, reduces metastasis, and increases survival in murine cancer models

Abstract In the tumor microenvironment, TIE2 expression on tissue macrophages, bone marrow derived TIE2-expressing monocytes (TEMs), osteoclasts, and vascular endothelial cells promotes tumor invasiveness, dissemination, and metastasis. Additionally, a subset of TIE2-expressing macrophages, located within specialized vascular structures known as tumor microenvironment for metastases (TMEMs), are linked to intravasation of cancer cells into circulation and dissemination to metastatic sites. Rebastinib is a picomolar inhibitor of TIE2 kinase, and exhibits an extraordinarily long off-rate from TIE2, measured to be over 24 hours in a cell-based assay. Herein, we examine the efficacy of rebastinib in the polyoma middle-T antigen (PyMT) syngeneic mouse breast cancer model. In this model, PyMT breast cancer cells are implanted in the mammary fat pad, and primary tumor growth leads to lung metastasis, which is known to be modulated by TEMs and TMEM vascular structures. We examined multiple dosing schedules of rebastinib in combination with anti-tubulin agents (ATAs). Rebastinib treatment in this model significantly ablated TEMs in the primary tumor stroma and caused a significant decrease in lung metastases. Furthermore, the combination of rebastinib with ATAs, even with once or twice weekly oral dosing of rebastinib, led to a significant further decrease in lung metastases compared to single-agent treatment with ATAs. Rebastinib also enhanced the activity of ATAs in reducing primary tumor growth and regrowth of tumor post-resection. TIE2 inhibition with targeted therapy represents a novel treatment approach for metastatic breast cancer and other cancers that rely on TEMs and TMEMs for growth and metastasis. As such, rebastinib has been selected for further clinical development in solid tumors with a Phase 1b trial being planned for 2014. This abstract is also presented as Poster A5. Citation Format: Daniel L. Flynn, Michael D. Kaufman, Cynthia B. Leary, Molly M. Hood, Wei-Ping Lu, Benjamin A. Turner, Scott C. Wise, Marc S. Rudoltz, Bryan D. Smith. Rebastinib, a selective TIE2 kinase inhibitor, decreases TIE2-expressing macrophages, reduces metastasis, and increases survival in murine cancer models. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR01. doi:10.1158/1538-7445.CHTME14-PR01

Abstract 1222: Establishment of a reliable metastasizing syngeneic breast cancer mouse model using orthotopically implanted 4T1 cells after several rounds of isolating and reimplanting lung metastases

Abstract Many human breast cancer cell lines have been implanted into immunodeficient mice in order to establish in vivo xenograft models, subcutaneous or orthotopic variants, in order to mimic this type of cancer as good as possible. However, especially the metastasising capabilities of these models turned out to be highly disappointing. In order to get more reliable models, breast cancer cells have often been implanted intravenously or intracardially rather than orthotopically. These models do work fine but resemble only limited parts of the complicated metastasis process. Therefore, a metastasis event originated from an orthotopically growing breast tumor seems more appropriate and displays an expanded view on the process of metastasis. In the model presented here, the murine brast cancer cell line 4T1 was transduced with fire fly luciferase and implanted orthotopically into the fat pad of female BALB/C mice. The metastasis rate in this model is known to be better than in the common human xenografts transplanted in mice, although still limited. In order to get higher metastasis rates we tried to improve this model by resecting and recultivating the metastases of the invaded lungs after orthotopic implantation of the 4T1 cells. Cultured cells were checked for in vitro luciferase activity in parallel with ex vivo analysis of the lung tissue part which was used for the isolation of the cells. After using this approach for several rounds, what we refer to as “subpopulationing”, we checked for increased metastasis properties of this newly isolated breast cancer cells. The new 4T1-M-ortho-luc cells were implanted ortotopically in parallel to the parental 4T1-luc cell line and we analysed 9 different organs ex vivo for luciferase activity. Whereas in the parental model only in lung tissue metastasis in all implanted mice (12/12) was detectable, the metastasis rate in the new model, generated by “subpopulationing”, bounced up to 4 organs with measurable metastasis in 100 % of the mice and in general to a much higher degree of metastasis in most other organs. Only in liver tissue no metastases could be detected in both models. Treatment of the mice with Doxorubicin clearly diminished tumor growth in both breast cancer models and can therefore serve as a potent positive control here. With this new 4T1-M-ortho cell line we have available a metastasing breast cancer cell line that shows reliable metastasis observed in several organs of the mouse when implanted orthotopically. This model is suitable for testing potential anti-meatstasizing compounds aginst breast cancer. In addition, since this model is established in immunocompetent mice, and is therefore also perfectly suited for approaches involving the immune system of breast cancer patients. Citation Format: Andreas Lingnau, Steffen Hoffmann, Sandra Moor, Cynthia Schaefer-Obodozie, Christoph Schaechtele. Establishment of a reliable metastasizing syngeneic breast cancer mouse model using orthotopically implanted 4T1 cells after several rounds of isolating and reimplanting lung metastases. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1222. doi:10.1158/1538-7445.AM2014-1222

Targeted Delivery of Curcumin to Tumors via PEG-Derivatized FTS-Based Micellar System

Curcumin and S-trans, trans-farnesylthiosalicylic acid (FTS) are two promising anticancer agents. In this study, we demonstrated that the two agents exerted significant synergy in antitumor activity in various types of cancer cells with combination indices ranging from 0.46 to 0.98 (a value of less than unity indicates synergism). We have further shown that synergistic-targeted co-delivery of the two agents can be achieved via formulating curcumin in polyethylene glycol (PEG)-derivatized FTS-based nanomicellar system. Curcumin formulated in PEG-FTS micelles had small size of around 20 nm. The nanomicellar curcumin demonstrated enhanced cytotoxicity towards several cancer cell lines in vitro. Intravenous application of curcumin-loaded micelle (20 mg kg(-1) curcumin) led to a significantly more effective inhibition of tumor growth in a syngeneic mouse breast cancer model (4T1.2) than curcumin formulated in Cremophor/EL (P < 0.05).

Abstract P4-15-13: Rebastinib in combination with paclitaxel ablates TIE2-expressing macrophages, reduces metastasis, and increases survival in the PyMT metastatic breast cancer model

Abstract In cancer models, TIE2 kinase plays an important role in angiogenesis, vasculogenesis, and tumor metastasis. TIE2 expression is largely restricted to vascular endothelial cells, tissue macrophages, and bone marrow derived TIE2-expressing monocytes (TEMs), which are proangiogenic, provasculogenic and enhance invasiveness. The hypoxic tumor environment engendered by damaging the vasculature with chemotherapy, radiation, or anti-angiogenic treatments leads to rebound tumor vascularization by an angiogenic switch from the VEGF pathway to the angiopoietin/TIE2 pathway. This leads to recruitment of provasculogenic TEMs from the bone marrow, leading to the growth of residual tumor cells and disease progression. Significantly, a subset of TIE2-expressing macrophages are located within specialized vascular structures known as tumor microenvironment for metastases (TMEMs). Recent observations have linked TIE2-expressing macrophages within TMEM structures to intravasation of cancer cells into circulation and subsequent dissemination to metastatic sites. We hypothesized that TIE2 inhibition should decrease migration and association of TEMs with blood vessels in the tumor stroma, therefore blocking their proangiogenic activity and leading to reduced tumor growth. TIE2 inhibition may also alter TMEM function, leading directly to a blockade of metastasis. Rebastinib is a picomolar inhibitor of TIE2 kinase, and exhibits an extraordinarily long off-rate from TIE2, measured to be over 24 hours in a cell-based assay. Herein, we examine the efficacy of rebastinib in the polyoma middle-T antigen (PyMT) syngeneic mouse breast cancer model. In this model, PyMT breast cancer cells are implanted in the mammary fat pad, and primary tumor growth leads to metastasis, which is known to be modulated by TEMs and TMEM vascular structures. We examined multiple dosing schedules of rebastinib in combination with paclitaxel. Rebastinib treatment in this model significantly ablated TEMs in the primary tumor stroma and caused a significant decrease in lung metastases (Table 1). Furthermore, the combination of rebastinib and paclitaxel led to a significant further decrease in lung metastases compared to treatment with paclitaxel or rebastinib alone. Rebastinib also enhanced the activity of paclitaxel in reducing primary tumor growth and regrowth of tumor post-resection. TIE2 inhibition with targeted therapy represents a novel treatment approach for metastatic breast cancer and other cancers that rely on TEMs and TMEMs for growth and metastasis. As such, rebastinib has been selected for further clinical development for treatment-refractory metastatic breast cancer, with a Phase 1b trial being planned for late 2013. Rebastinib reduces lung metastases in the PyMT breast cancer modelStudynTreatmentLung Metastases (% of Control)110Vehicle100%110Paclitaxel 10 mg/kg Q5D36%110Rebastinib 10 mg/kg BID28%110Rebastinib 10 mg/kg BID + Paclitaxel7%210Vehicle100%210Paclitaxel 10 mg/kg Q5D51%210Rebastinib 10 mg/kg QD + Paclitaxel21%33Vehicle100%33Paclitaxel 10 mg/kg Q5D58%33Rebastinib 10 mg/kg twice/week + Paclitaxel28% Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-13.