Murine Triple-negative Breast Cancer Model Research Articles

Abstract PD2-08: Targeting of eIF4A1 along with anti-PD-L1 therapy limits lung metastases efficaciously in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype of metastatic breast cancer with poor clinical prognosis. Currently used neoadjuvant chemotherapy for TNBC, such as taxanes, and/or anthracyclines demonstrate an initial treatment response but is often followed by drug resistance, tumor relapse, and high proclivity to develop metastases. There is an unmet need to develop effective combination therapies against metastatic TNBC (mTNBC). eIF4A1 is an mRNA helicase that help translate many oncogenic mRNAs including PD-L1 (through STAT1 translational regulation) that are involved in tumor growth, chemoresistance and metastases. Our prior studies pointed out that eIF4A1 is intricately linked to cancer stemness and drug resistance in TNBC. Emerging evidence has revealed that the tumor immune microenvironment (TIME) of TNBC do consist of mononuclear cells and lymphocytes. Combining chemotherapy with immune checkpoint inhibition had demonstrated a significant benefit for high-risk TNBC patients. In this study, we examine the efficacy of targeting eIF4A1 along with anti-PD-L1 therapy as an effective therapeutic strategy to limit cancer stemness, primary tumor progression and metastasis in an immunocompetent murine model of TNBC. Approach: Initially, we examined the human TNBC biospecimens and found that eIF4A1 protein level is upregulated in drug-resistant cases. STAT1 is an upstream regulator of PD-L1 and a downstream effector of eIF4A1. The protein level of STAT1 was significantly elevated as assessed by immunoblotting of human tumor lysates. In human TNBC cells, when eIF4A1 is pharmacologically or genetically targeted, PD-L1 was downregulated at the protein level. Based on this premise, we targeted eIF4A1 with the small molecule inhibitor, Rocaglamide A (RocA), along with anti-PD-L1 immunotherapy in orthotopically implanted 4T1 mouse TNBC tumors in BALB/c mice. The treatment arms were 1) Control (vehicle for RocA + control IgG) 2) RocA 3) RocA + anti-PD-L1 antibodies 4) Control IgG only 5) Anti-PD-L1 antibodies only with 10 mice in each group. Results: The combination treatment comprising of RocA and anti-PD-L1 antibodies worked out efficaciously as the primary tumor volume, wet tumor weight, bioluminescent tumor imaging and quantitation were significantly lower than the combination vehicle control. Furthermore, metastasis to the lungs were significantly impaired (1 out of 10 mice only had metastasis and that too it was small). On the contrary, ‘RocA single arm’ group had 6 out of 10 mice with lung metastasis. The lung metastases were observed in 4 out of 10 mice in the PD-L1 single arm and 6 out of 10 mice in control IgG single arm group. The RocA was tolerated well at 0.6 mg/kg with no significant change in the body weight and the liver and kidney function test values were within the normal range. Importantly, the combination treatment with RocA and PD-L1 was also tolerated well. Conclusions: The combination therapy involving RocA and PD-L1 had the significant impact in reducing lung metastases and may pave the way for phase I/II clinical trials. Citation Format: Dharmindra Dulal, Andrew Boring, Dayanidhi Raman. Targeting of eIF4A1 along with anti-PD-L1 therapy limits lung metastases efficaciously in triple-negative breast cancer [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 PD2-08.

Mathematical Model of Triple-Negative Breast Cancer in Response to Combination Chemotherapies.

Triple-negative breast cancer (TNBC) is a heterogenous disease that is defined by its lack of targetable receptors, thus limiting treatment options and resulting in higher rates of metastasis and recurrence. Combination chemotherapy treatments, which inhibit tumor cell proliferation and regeneration, are a major component of standard-of-care treatment of TNBC. In this manuscript, we build a coupled ordinary differential equation model of TNBC with compartments that represent tumor proliferation, necrosis, apoptosis, and immune response to computationally describe the biological tumor affect to a combination of chemotherapies, doxorubicin (DRB) and paclitaxel (PTX). This model is parameterized using longitudinal [18F]-fluorothymidine positron emission tomography (FLT-PET) imaging data which allows for a noninvasive molecular imaging approach to quantify the tumor proliferation and tumor volume measurements for two murine models of TNBC. Animal models include a human cell line xenograft model, MDA-MB-231, and a syngeneic 4T1 mammary carcinoma model. The mathematical models are parameterized and the percent necrosis at the end time point is predicted and validated using histological hematoxylin and eosin (H&E) data. Global Sobol' sensitivity analysis is conducted to further understand the role each parameter plays in the model's goodness of fit to the data. In both the MDA-MB-231 and the 4T1 tumor models, the designed mathematical model can accurately describe both tumor volume changes and final necrosis volume. This can give insight into the ordering, dosing, and timing of DRB and PTX treatment. More importantly, this model can also give insight into future novel combinations of therapies and how the immune system plays a role in therapeutic response to TNBC, due to its calibration to two types of TNBC murine models.

3′-epi-12β-hydroxyfroside-mediated autophagy degradation of RIPK1/RIPK3 necrosomes leads to anergy of immunogenic cell death in triple-negative breast cancer cells

Increasing studies have suggested that some cardiac glycosides, such as conventional digoxin (DIG) and digitoxin, can induce immunogenic cell death (ICD) in various tumors. We previously found that 3′-epi-12β-hydroxyfroside (HyFS), a novel cardenolide compound isolated by our group, could induce cytoprotective autophagy through inactivation of the Akt/mTOR pathway. However, whether HyFS can induce ICD remains unknown. In this study, we extend our work to further investigate whether HyFS could induce both autophagy and ICD, and we investigated the relationship between autophagy and ICD in three TNBC cell lines. Unexpectedly, compared to DIG, we found that HyFS could induce complete autophagy flux but not ICD in three human triple-negative breast cancer (TNBC) cell lines and one murine TNBC model. Inhibition of HyFS-induced autophagy resulted in the production of ICD in TNBC MDA-MB-231, MDA-MB-436, and HCC38 cells. A further mechanism study showed that formation of RIPK1/RIPK3 necrosomes was necessary for ICD induction in DIG-treated TNBC cells, while HyFS treatment led to receptor-interacting serine-threonine kinase (RIPK)1/3 necrosome degradation via an autophagy process. Additionally, inhibition of HyFS-induced autophagy by the autophagy inhibitor chloroquine resulted in the reoccurrence of ICD and reversion of the tumor microenvironment, leading to more significant antitumor effects in immunocompetent mice than in immunodeficient mice. These findings indicate that HyFS-mediated autophagic degradation of RIPK1/RIPK3 necrosomes leads to inactivation of ICD in TNBC cells. Moreover, combined treatment with HyFS and an autophagy inhibitor may enhance the antitumor activities, suggesting an alternative therapeutic for TNBC treatment.

Enhanced Therapeutic Effect of Optimized Melittin-dKLA, a Peptide Agent Targeting M2-like Tumor-Associated Macrophages in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is characterized by a high possibility of metastasis. M2-like tumor-associated macrophages (TAMs) are the main components of the tumor microenvironment (TME) and play a key role in TNBC metastasis. Therefore, TAMs may be a potential target for reducing TNBC metastasis. Melittin-dKLA, a peptide composed of fused melittin and pro-apoptotic peptide d(KLAKLAK)2 (dKLA), showed a potent therapeutic effect against cancers by depleting TAMs. However, melittin has a strong adverse hemolytic effect. Hence, we attempted to improve the therapeutic potential of melittin-dKLA by reducing toxicity and increasing stability. Nine truncated melittin fragments were synthesized and examined. Of the nine peptides, the melittin-dKLA8-26 showed the best binding properties to M2 macrophages and discriminated M0/M1/M2. All fragments, except melittin, lost their hemolytic effects. To increase the stability of the peptide, melittin-dKLA8-26 fragment was conjugated with PEGylation at the amino terminus and was named PEG-melittin-dKLA8-26. This final drug candidate was assessed in vivo in a murine TNBC model and showed superior effects on tumor growth, survival rates, and lung metastasis compared with the previously used melittin-dKLA. Taken together, our study showed that the novel PEG-melittin-dKLA8-26 possesses potential as a new drug for treating TNBC and TNBC-mediated metastasis by targeting TAMs.

Anti-TGF-β/PD-L1 bispecific antibody promotes T cell infiltration and exhibits enhanced antitumor activity in triple-negative breast cancer

BackgroundAgents blocking programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) have been approved for triple-negative breast cancer (TNBC). However, the response rate of anti-PD-1/PD-L1 is still unsatisfactory, partly due to...

DNA minicircles as novel STAT3 decoy oligodeoxynucleotides endowed with anticancer activity in triple-negative breast cancer

Decoy technology is a versatile and specific DNA oligonucleotide-based targeting strategy of pathogenic transcription factors (TFs). Chemical modifications of linear decoy oligonucleotides have been made to decrease nuclease sensitivity because of the presence of free ends but at the cost of new limitations that affect their use as therapeutic drugs. Although a short DNA minicircle is a phosphodiester nucleic acid without free ends, its potential therapeutic activity as a TF decoy oligonucleotide has not yet been investigated. Here we describe the in vitro and in vivo activity of formulated 95-bp minicircles bearing one or several STAT3 binding sequences in triple-negative breast cancer (TNBC). Minicircles bearing one STAT3 binding site interacted specifically with the active form of STAT3 and inhibited proliferation, induced apoptosis, slowed down cell cycle progression, and decreased STAT3 target gene expression in human and murine TNBC cells. Intratumoral injection of STAT3 minicircles inhibited tumor growth and metastasis in a murine model of TNBC. Increasing the number of STAT3 binding sites resulted in improved anticancer activity, opening the way for a TF multitargeting strategy. Our data provide the first demonstration of minicircles acting as STAT3 decoys and show that they could be an effective therapeutic drug for TNBC treatment.

Abstract 1627: Metabolic links between obesity and ferroptosis in a murine model of breast cancer

Abstract Background Obesity is an established risk factor for post-menopausal triple negative breast cancer (TNBC). Multiple aspects of fatty acid metabolism, including fatty acid synthesis, are upregulated in white adipose tissue during obesity development. The enzyme pyruvate carboxylase (PC), a supportive player in fatty acid synthesis, is relied upon for metastasis of murine TNBC particularly in models of obesity- further elucidating the importance of fatty acid metabolism to breast cancer progression in those with obesity. Ferroptosis, a method of cell death induced by peroxidation of phospholipid fatty acyl chains, is a recently discovered and rapidly developing mechanistic target in multiple cancer types. While several cancers, including breast cancer, are sensitive to ferroptosis induced by different mechanisms, the metabolic vulnerabilities and consequent therapeutic potential of ferroptosis are just beginning to be explored. This study aims to determine whether obesity-driven reprogramming of fatty acid metabolism exacerbates breast cancer progression via altered sensitivity to ferroptosis Methods The relationship between obesity, breast cancer and lipid peroxidation was investigated in vitro via proliferation assays, qPCR, Western blot and flow cytometry using multiple murine models of breast cancer treated with erastin and RSL3. PC was suppressed using shRNA introduced via lentiviral transduction. In vivo tumor growth in lean and obese mice was determined following treatment with erastin, RSL3, or a vehicle control. Results In vitro data reveals that TNBC cells are highly sensitive to treatment with the ferroptosis-inducing molecules erastin and RSL3. Erastin treatment transcriptionally induces PC expression in several murine models of TNBC in vitro. Suppression of PC at baseline (without ferroptosis induction) is sufficient to induce transcription of glutathione peroxidase-4 (a critical regulator of ferroptosis). While Affymetrix array data of untreated metastatic M-WntLung primary tumors showed a decrease in PC and associated genes in tumors from obese mice relative to lean mice, treatment with erastin increased PC expression specifically in tumors from obese mice. Tumors from obese mice relative to control mice have an altered response to treatment with the ferroptosis inducing molecules erastin and RSL3. Conclusion These data indicate that obesity-associated dysregulation of lipid metabolism and regulation of PC may be key determinants of sensitivity to induction of ferroptosis. Future work will delineate the relationship between breast cancer, ferroptosis, and fatty acid metabolism utilizing in vivo models to determine whether ferroptosis-specific mechanisms are exacerbated with PC suppression to further impede obesity-exacerbated tumor growth. Citation Format: Emily Devericks, Michael F. Coleman, Hannah Malian, Violet Kiesel, Dorothy Teegarden, Stephen D. Hursting. Metabolic links between obesity and ferroptosis in a murine model of breast cancer [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 1627.

Combination of OX40 Co-Stimulation, Radiotherapy, and PD-1 Inhibition in a Syngeneic Murine Triple-Negative Breast Cancer Model.

Simple SummaryThis experimental study was designed in order to investigate the efficacy of the triple combination of radiation (SBRT), PD-1 blockade, and OX40 co-stimulation in a syngeneic murine model using ‘immunologically cold’ triple-negative breast cancer cells. SBRT can induce immunogenic tumor cell deaths and act as an in situ vaccine while OX40 signaling has been shown to improve anticancer immunity combined with PD-1 inhibition via multiple preclinical studies. In our study, triple combination therapy significantly improved primary/abscopal tumor control and reduced lung metastases compared to single or dual therapies. This was found to be through an increased ratio of CD8+ T cells to regulatory T cells and a reduced proportion of exhausted T cells in the tumor microenvironment.Immune checkpoint inhibitors have been successful in a wide range of tumor types but still have limited efficacy in immunologically cold tumors, such as breast cancers. We hypothesized that the combination of agonistic anti-OX40 (α-OX40) co-stimulation, PD-1 blockade, and radiotherapy would improve the therapeutic efficacy of the immune checkpoint blockade in a syngeneic murine triple-negative breast cancer model. Murine triple-negative breast cancer cells (4T1) were grown in immune-competent BALB/c mice, and tumors were irradiated with 24 Gy in three fractions. PD-1 blockade and α-OX40 were administered five times every other day. Flow cytometric analyses and immunohistochemistry were used to monitor subsequent changes in the immune cell repertoire. The combination of α-OX40, radiotherapy, and PD-1 blockade significantly improved primary tumor control, abscopal effects, and long-term survival beyond 2 months (60%). In the tumor microenvironment, the ratio of CD8+ T cells to CD4 + FOXP3+ regulatory T cells was significantly elevated and exhausted CD8+ T cells (PD-1+, CTLA-4+, TIM-3+, or LAG-3+ cells) were significantly reduced in the triple combination group. Systemically, α-OX40 co-stimulation and radiation significantly increased the CD103+ dendritic cell response in the spleen and plasma IFN-γ, respectively. Together, our results suggest that the combination of α-OX40 co-stimulation and radiation is a viable approach to overcome therapeutic resistance to PD-1 blockade in immunologically cold tumors, such as triple-negative breast cancer.

“X”ploring the multifaceted functions of NLRX1 in triple-negative breast cancer

Abstract Breast cancer is a devastatingly common and deadly malignancy, and despite improved survival resulting from improved detection and treatment, triple-negative breast cancer (TNBC) remains difficult to treat. While historically studied in the context of infectious diseases, a multifaceted anti-inflammatory pattern recognition receptor called NLRX1 has recently been investigated in several types of cancers due to its involvement in several pathways significant to tumorigenesis and disease progression. This project seeks to define the role of NLRX1 in TNBC. To achieve this, we generated novel Nlrx1−/−(KO) mice compatible with the widely-used 4T1 murine model of TNBC. We also generated 4T1 cells that either overexpress or knockdown NLRX1, as well as their respective controls (4T1OE, 4T1OE-CTL, 4T1KD, 4T1KD-CTL). KO and wild-type (WT) mice were then injected with unaltered 4T1 cells or one of the four altered 4T1 cell lines. We then quantified tumor growth, final tumor volume, and lung metastasis as a measure of disease burden. This in vivo work demonstrated that NLRX1 is protective against disease burden when expressed in healthy host tissues but is problematic when overexpressed by 4T1 cells. Ex vivo and in vitro experiments then revealed a pro-migratory phenotype in 4T1OE cells, likely as the result of altered TGF-β signaling and/or the PI3K-Akt-mTOR pathway as a means to impact epithelial-mesenchymal transition. Combined, we believe the function of NLRX1 in TNBC depends on the cell in which it is expressed (i.e. healthy vs cancerous), and that this function may ultimately be a result of altered epithelial-mesenchymal transition pathways.

Reduction of Metastasis via Epigenetic Modulation in a Murine Model of Metastatic Triple Negative Breast Cancer (TNBC).

Simple SummaryWe investigate the use of the small molecule epigenetic modulator 4SC-202 as a potential cancer therapeutic or adjuvant for Triple Negative Breast Cancer (TNBC). Epigenetic modulation involves alteration of the cellular phenotype without altering the genotype. Here, we investigate how 4SC-202 affects tumor growth, the ability of tumor cells to migrate, and the growth of tumors in vivo. This study demonstrates that 4SC-202 kills tumor cells, reduces their ability to migrate, and decreases metastasis of the tumor cells and tumor burden in a highly metastatic murine model of TNBC. These preclinical studies provide critical information on the potential efficacy of 4SC-202 as a potential therapeutic or adjuvant for TNBC.This study investigates the effects of a dual selective Class I histone deacetylase (HDAC)/lysine-specific histone demethylase 1A (LSD1) inhibitor known as 4SC-202 (Domatinostat) on tumor growth and metastasis in a highly metastatic murine model of Triple Negative Breast Cancer (TNBC). 4SC-202 is cytotoxic and cytostatic to the TNBC murine cell line 4T1 and the human TNBC cell line MDA-MB-231; the drug does not kill the normal breast epithelial cell line MCF10A. Furthermore, 4SC-202 reduces cancer cell migration. In vivo studies conducted in the syngeneic 4T1 model, which closely mimics human TNBC in terms of sites of metastasis, reveal reduced tumor burden and lung metastasis. The mechanism of action of 4SC-202 may involve effects on cancer stem cells (CSC) which can self-renew and form metastatic lesions. Approximately 5% of the total 4T1 cell population grown in three-dimensional scaffolds had a distinct CD44high/CD24low CSC profile which decreased after treatment. Bulk transcriptome (RNA) sequencing analyses of 4T1 tumors reveal changes in metastasis-related pathways in 4SC-202-treated tumors, including changes to expression levels of genes implicated in cell migration and cell motility. In summary, 4SC-202 treatment of tumors from a highly metastatic murine model of TNBC reduces metastasis and warrants further preclinical studies.

Neoadjuvant Acoustic Immune Priming with Low-Intensity Focused Ultrasound and Doxil for in Situ Tumor Vaccination in a Murine Triple-Negative Breast Cancer Model

Keisling, Shannon M. MD, MPH; Singh, Saurabh PhD; Chavez, Claudia Gutierrez MSc; Kumar, Mayank PhD; Karunakaran, Chandra PhD; Guha, Chandan MD, PhD Author Information

Comparative colloidal stability, antitumor efficacy, and immunosuppressive effect of commercial paclitaxel nanoformulations

BackgroundStandard chemotherapy with taxanes, such as paclitaxel (PTX), remains the mainstay of systemic treatment of triple-negative breast cancer. Nanotechnology-based formulations have gradually replaced PTX injection and are widely used in China. However, no studies have compared the colloidal stability, antitumor efficacy, and safety of commercial PTX nanoformulations. Additionally, the desire to evaluate preclinical antitumor efficacy in human-derived tumor cells led to the widespread application of immunodeficient mouse models that likely contributed to the neglect of nanomedicines-immune system interactions. The present study investigated the colloidal stability, antitumor efficacy and safety, and nanomedicines-host immune system interactions of PTX nanoformulations. A further comparative analysis was performed to evaluate the clinical potential.ResultsCompared with liposome, PTX emulsion and PTX nanoparticle exhibited favorable colloidal stability. PTX emulsion was superior in inducing apoptosis and had a more pronounced inhibitory effect on 4T1-tumor spheroids compared with PTX liposome and PTX nanoparticle. Although PTX emulsion exhibited superior in vitro antitumor effect, no significant differences in the in vivo antitumor efficacy were found among the three types of PTX nanoformulations in an immunocompetent orthotopic 4T1 murine triple-negative breast cancer model. All PTX nanoformulations at maximum tolerated dose (MTD) induced lymphopenia and immunosuppression, as evidenced by the reduction of T cell subpopulations and inhibition of the dendritic cells maturation.ConclusionsThe MTD PTX nanomedicines-induced lymphopenia and immunosuppression may weaken the lymphocyte-mediated antitumor cellular immune response and partly account for the lack of differences in the in vivo antitumor outcomes of PTX nanoformulations. Understanding of what impacts PTX nanomedicines has on the immune system may be critical to improve the design and conduct of translational research of PTX nanomedicines in monotherapy or combination therapy with immunotherapy.Graphic abstract

PI3Kαδ Inhibitor Combined With Radiation Enhances the Antitumor Immune Effect of Anti-PD1 in a Syngeneic Murine Triple-Negative Breast Cancer Model

The poor response of breast cancer to immune checkpoint blockade might result from low immunogenicity and the immune-suppressive tumor microenvironment. We hypothesized that in situ tumor vaccination via radiation therapy (RT) and suppression of immune tolerance via phosphoinositide 3-kinase δ (PI3Kδ) inhibition would enhance the efficacy of immune checkpoint blockade. 4T1 murine breast cancer cells were grown in both immune-competent and -deficient BALB/c mice, and tumors were irradiated with 24 Gy in 3 fractions. A PD-1 blockade and a PI3Kαδ inhibitor were then administered every other day for 2 weeks. Fluorescence-activated cell sorting and immunohistochemistry served to monitor subsequent changes in immune cell repertoire. The triple combination of RT, PD-1 blockade, and PI3Kαδ inhibitor significantly delayed tumor growth. The immune-deficient syngeneic 4T1 murine tumor model failed to show this tumor growth delay. Use of RT and PI3Kαδ inhibitor increased the proportions of CD8+ T cells; PI3Kαδ inhibitor led to a decrease in regulatory T cells and polymorphonuclear myeloid-derived suppressor cells. The triple combination resulted in a remarkable increase in cytotoxic CD8+ T cells, suggesting a prominent immune-modulatory effect. The abscopal effect was most prominent in the triple-combination therapy group, and it correlated with splenic CD8+ T cell accumulation. These findings collectively indicate that combining RT, PI3Kαδ inhibitor, and PD-1 blockade could be a viable approach, helping to overcome the therapeutic resistance of immunologically cold tumors, such as breast cancer, with an immunosuppressive tumor microenvironment.

Alfalfa mosaic virus nanoparticles-based in situ vaccination induces antitumor immune responses in breast cancer model.

Background: Preclinical and clinical studies show that local and systemic antitumor efficacy is achievable by in situ vaccination (ISV)using plant virus nanoparticles in which immunostimulatory reagents are directly administered into the tumor rather than systemically. Aim: To investigate a minimally studied plant virus nanoparticle, alfalfa mosaic virus (AMV), for ISVtreatment of 4T1, the very aggressive and metastatic murine triple-negative breast cancer model. Materials& methods: AMV nanoparticles were propagated and characterized. Their treatment impact on in vivo tumors were analyzed using determination of inherent immunogenicity, cytokine analysis, western blotting analysis and immunohistochemistry methodologies. Results: AMV used as an ISV significantly slowed down tumor progression and prolonged survival through immune mechanisms (p<0.001). Conclusion: Mechanistic studies show that ISV with AMV increases costimulatory molecules, inflammatory cytokinesand immune effector cell infiltration and downregulates immune-suppressive molecules.

Integrin αvβ6–TGFβ–SOX4 Pathway Drives Immune Evasion in Triple-Negative Breast Cancer

Cancer immunotherapy shows limited efficacy against many solid tumors that originate from epithelial tissues, including triple-negative breast cancer (TNBC). We identify the SOX4 transcription factor as an important resistance mechanism to Tcell-mediated cytotoxicity for TNBC cells. Mechanistic studies demonstrate that inactivation of SOX4 in tumor cells increases the expression of genes in a number of innate and adaptive immune pathways important for protective tumor immunity. Expression of SOX4 is regulated by the integrin αvβ6 receptor on the surface of tumor cells, which activates TGFβ from a latent precursor. An integrin αvβ6/8-blocking monoclonal antibody (mAb) inhibits SOX4 expression and sensitizes TNBC cells to cytotoxic Tcells. This integrin mAb induces a substantial survival benefit in highly metastatic murine TNBC models poorly responsive to PD-1 blockade. Targeting of the integrin αvβ6-TGFβ-SOX4 pathway therefore provides therapeutic opportunities for TNBC and other highly aggressive human cancers of epithelial origin.

Targeting CXCL12-CXCR4 Signaling Enhances Immune Checkpoint Blockade Therapy Against Triple Negative Breast Cancer

Insufficient T cell infiltration in triple-negative breast cancer (TNBC) has limited its response rate to immune checkpoint blockade (ICB) therapies and motivated the development of immunostimulatory approaches to enhance the ICB therapy. CXCR4 is a chemokine receptor highly upregulated both on cell surface and cytoplasm in tumor tissues. Activating CXCR4 has been associated with increased immunosuppression in the tumor microenvironment. Here, we developed a CXCR4-targeted liposomal formulation (Liposomal-AMD3100) to enhance therapeutic efficacy of AMD3100, a CXCR4 antagonist. Particularly, AMD3100 is not only encapsulated into the liposome but coated on the surface of the formulation to serve as a targeting moiety and a dual blocker capable of inhibiting CXCR4 activation extracellularly and intracellularly. The Liposomal-AMD3100 remodeled both immune and stromal microenvironment more efficiently compared with free AMD3100, indicating better pharmacodynamic profile of AMD3100 achieved by liposomal formulation. The combination of anti-PD-L1 with Liposomal-AMD3100 formulation exhibited an increased antitumor effect and prolonged survival time compared with monotherapies in a murine TNBC model (4T1). This work proves that immune activation via liposomal delivery of CXCR4 inhibitors has a great potential to expand ICB therapies to originally ICB-insensitive cancer types.

Abstract 6198: Regulation of cycloxygenase-2 in the tumor micro-environment improves radiation and immunotherapy

Abstract In breast cancer (BC), the presence of tumor infiltrating lymphocytes is associated with improved survival. A recent study showed that increased CD8 cells and Th17 cells are specifically associated with triple negative breast cancer (TNBC) patients, a highly aggressive subclass of breast cancer. However, they undergo functional reprogramming in the tumor micro-environment(TME) evident from decreased IFN-γ;; and granzyme B. These immune escape mechanisms contribute to inability of the immune system to control tumor progression. Thus modulation of TME is necessary to effectively target the tumor. Radiation therapy (RT) is commonly used in more than 60% of cancer patients including BC. Focal radiation limits systemic side effects commonly associated with chemotherapy and acts as immune modulator. Eventually the tumor comes out of the growth delay and tends to show more aggressive phenotype. We found that RT induced inflammation associated biomarkers nitric oxide synthase2 (NOS2) and cycloxygenase2 (COX2) in the TME, specifically in the tumor cells.We previously showed that co-expression of pro-inflammatory enzymes NOS2 and COX2is a powerful prognostic indicator of poor outcome (HR=21) among ER-patients which in turn drive major oncogenic pathways. Immunotherapy, on the other hand, is being used as a standard of care in lung cancer. However, currently there is no approved immunotherapy available for BC patients, early data from several ongoing clinical trials show activity in various subclasses of BC including TNBC. It is reported that PD-L1 is high in 20% of TNBCs and COX2 may be involved in its regulation in tumor-infiltrating myeloid cells. This led us to hypothesize that modulation of inflammation associated biomarkers in the TME would increase the efficacy of RT and immunotherapy by amplifying anti-tumor immunity. We investigated the effect of NOS2 or COX2 inhibition using commercially available inhibitor on radiation and αPD-L1 induced tumor growth delay and lung metastases in murine model of TNBC using 4T1 cell line implanted in flank of Balbc mice. Change in immune cell populations in the TME was investigated using confocal microscopy, CO-Detection by indEXing (CODEX) technology and flow-cytometry. We also measured the levels of inflammation associated cytokines in serum. We demonstrated that co-treatment with COX2 inhibitor led to tumor growth delay and reduced metastases compared to conventional therapy by changing the TME to support tumor clearance. Citation Format: Debashree Basudhar, Veena Somasundaram, David A. Scheiblin, Noemi Kedei, Robert Y. Cheng, Lisa A. Ridnour, Daniel W. McVicar, Stephen Lockett, David A. Wink. Regulation of cycloxygenase-2 in the tumor micro-environment improves radiation and immunotherapy [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 6198.

Abstract 1761: Examination of therapeutic potential of epigenetic modulation by dual HDAC-LSD1 inhibition in murine models of triple negative breast cancer (TNBC)

Abstract This study examines the effects of the epigenetic modulator 4SC-202, a dual histone deacetylase (HDAC) /lysine-specific histone demethylase 1A (LSD1) small molecule inhibitor, on tumor growth and metastasis in a mouse model of breast cancer. 4SC-202 differs from clinically available HDAC inhibitors such as Vorinostat (SAHA) by specifically targeting class I HDACs including HDAC1, HDAC2, and HDAC3 and the histone demethylase LSD1. The hypothesis to be examined is that epigenetic modulation via dual inhibition of HDAC1 and LSD1 improves disease control in breast cancer. In vitro data indicates that 4SC-202 is significantly cytotoxic and cytostatic to the highly metastatic murine Triple Negative Breast Cancer (TNBC) murine model 4T1 and the human TNBC cell line MDA-MB-231 and reduces cancer cell migration. In vivo studies conducted in the syngeneic 4T1 model which closely mimics human TNBC in terms of sites of metastasis, show increased necrosis in 4T1 tumors, reduced tumor burden in vivo as compared to Vorinostat treatment, and significantly reduced metastasis. Furthermore, multiple studies in different cell types indicate that the mechanism of action of 4SC-202 may involve effects on cancer stem cells, which are able to self-renew and form metastatic lesions. It has been shown that a marker of cancer stem cells (CSC's) in breast cancer is high aldehyde dehydrogenase (ALDH) activity and that ALDH high cells mediate metastasis. In the 4T1 syngeneic mouse model of breast cancer, inhibition of ALDH activity suppresses stem-like cell properties. Preliminary data suggests 4SC-202 substantially reduces the ALDH high 4T1 cancer stem population. Transcriptome bulk RNA -sequencing analysis from 4T1 tumors reveal changes in metastatic related pathways in 4SC-202 treated tumors, including significant changes to the expression levels of genes implicated in focal adhesion and ECM-receptor interaction pathways. Furthermore, 4SC-202 appears to alter expression of cytokines which serve as chemokines, potentially leading to increased expression of tumor infiltrating lymphocytes. In summary, treatment of a highly metastatic murine model of TNBC with 4SC-202 significantly reduces metastasis, and warrants further preclinical studies in conjunction with standard of care chemotherapy treatment. Citation Format: Shanta M. Messerli, Mariah M. Hoffman, Jessica S. Zylla, Etienne Gnimpieba, Keith W. Miskimins. Examination of therapeutic potential of epigenetic modulation by dual HDAC-LSD1 inhibition in murine models of triple negative breast cancer (TNBC) [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 1761.

Id Proteins Promote a Cancer Stem Cell Phenotype in Mouse Models of Triple Negative Breast Cancer via Negative Regulation of Robo1.

Breast cancers display phenotypic and functional heterogeneity and several lines of evidence support the existence of cancer stem cells (CSCs) in certain breast cancers, a minor population of cells capable of tumor initiation and metastatic dissemination. Identifying factors that regulate the CSC phenotype is therefore important for developing strategies to treat metastatic disease. The Inhibitor of Differentiation Protein 1 (Id1) and its closely related family member Inhibitor of Differentiation 3 (Id3) (collectively termed Id) are expressed by a diversity of stem cells and are required for metastatic dissemination in experimental models of breast cancer. In this study, we show that ID1 is expressed in rare neoplastic cells within ER-negative breast cancers. To address the function of Id1 expressing cells within tumors, we developed independent murine models of Triple Negative Breast Cancer (TNBC) in which a genetic reporter permitted the prospective isolation of Id1+ cells. Id1+ cells are enriched for self-renewal in tumorsphere assays in vitro and for tumor initiation in vivo. Conversely, depletion of Id1 and Id3 in the 4T1 murine model of TNBC demonstrates that Id1/3 are required for cell proliferation and self-renewal in vitro, as well as primary tumor growth and metastatic colonization of the lung in vivo. Using combined bioinformatic analysis, we have defined a novel mechanism of Id protein function via negative regulation of the Roundabout Axon Guidance Receptor Homolog 1 (Robo1) leading to activation of a Myc transcriptional programme.

Interleukin-34 contributes to poor prognosis in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a subtype characterized by the absence of therapeutic targets. It shows rapid progression, higher relapse, and poor prognosis, so the establishment of an effective therapeutic target is required. We focused on interleukin-34 (IL-34) that is a novel cytokine relating to inflammation and tumorigenesis. It has been reported that IL-34 correlates with poor prognosis of various cancers. In this study, we evaluated the relationship of IL-34 and prognosis in TNBC using human clinical information and mice model. We found that IL-34 was highly expressed in TNBC, and the survival rate in TNBC was significantly lower in patients with high IL-34 expression. Furthermore, multivariate analysis revealed that IL-34 independently affects prognosis. In murine TNBC model, IL-34 deficiency in tumor cells decreased in vivo tumor growth and increased inflammatory cytokine production from macrophages. These results suggest that tumor-derived IL-34 creates a favorable environment for TNBC cells. Thus, we showed a novel pathological role of IL-34 in TNBC and the potential of IL-34 as a therapeutic target for it.