Triple-negative Breast Cancer Mouse Research Articles

MYC promotes immune-suppression in triple-negative breast cancer via inhibition of interferon signaling

The limited efficacy of immune checkpoint inhibitor treatment in triple-negative breast cancer (TNBC) patients is attributed to sparse or unresponsive tumor-infiltrating lymphocytes, but the mechanisms that lead to a therapy resistant tumor immune microenvironment are incompletely known. Here we show a strong correlation between MYC expression and loss of immune signatures in human TNBC. In mouse models of TNBC proficient or deficient of breast cancer type 1 susceptibility gene (BRCA1), MYC overexpression dramatically decreases lymphocyte infiltration in tumors, along with immune signature remodelling. MYC-mediated suppression of inflammatory signalling induced by BRCA1/2 inactivation is confirmed in human TNBC cell lines. Moreover, MYC overexpression prevents the recruitment and activation of lymphocytes in both human and mouse TNBC co-culture models. Chromatin-immunoprecipitation-sequencing reveals that MYC, together with its co-repressor MIZ1, directly binds promoters of multiple interferon-signalling genes, resulting in their downregulation. MYC overexpression thus counters tumor growth inhibition by a Stimulator of Interferon Genes (STING) agonist via suppressing induction of interferon signalling. Together, our data reveal that MYC suppresses innate immunity and facilitates tumor immune escape, explaining the poor immunogenicity of MYC-overexpressing TNBCs.

Surgical wounding enhances pro-tumor macrophage responses and accelerates tumor growth and lung metastasis in a triple negative breast cancer mouse model.

Approximately one-third of all breast cancer mortality results from metastatic recurrence after initial success of surgery and/or therapy. Although primary tumor removal is widely accepted as beneficial, it has long been suspected that surgery itself contributes to accelerated metastatic recurrence. We investigated surgical wounding's impact on tumor progression and lung metastasis in a murine model of triple negative breast cancer (TNBC). Ten-week-old female mice were inoculated with 4T1 cells (week 0) and were either subjected to a 2cm long cutaneous contralateral incision (wounded) or control (non-wounded) on week 2 and monitored for 3 weeks (week 5). Mice with surgical wounding displayed significantly accelerated tumor growth observable as early as 1-week post wounding. This was confirmed by increased tumor volume and tumor weight, post-mortem. Further, surgical wounding increased metastasis to the lungs, as detected by IVIS imaging, in vivo and ex vivo (week 5). As expected then, wounded mice displayed decreased apoptosis and increased proliferation in both the primary tumor and in the lungs. Flow cytometry revealed that primary tumors from wounded mice exhibited increased tumor associated macrophages and specifically M2-like macrophages, which are important in promoting tumor development, maintenance, and metastasis. Immunofluorescence staining and gene expression data further confirms an increase in macrophages in both the primary tumor and the lungs of wounded mice. Our data suggests that surgical wounding accelerates tumor progression and lung metastasis in a mouse model of TNBC, which is likely mediated, at least in part by an increase in macrophages.

Single-cell RNA-sequencing uncovers compound kushen injection synergistically improves the efficacy of chemotherapy by modulating the tumor environment of breast cancer.

Due to lack of enough specific targets and the immunosuppressive tumor microenvironment (TME) of triple-negative breast cancer (TNBC), TNBC patients often cannot benefit from a single treatment option. This study aims to explore the regulatory effects of Compound kushen injection (CKI) plus chemotherapy on the TME of TNBC from a single cell level. A mouse TNBC model in BALB/c mice was established to evaluate the antitumor efficacy and toxicity of CKI combined with chemotherapy. Flow cytometry was used to observe the influence of CKI on the lymphocyte populations in the tumor bearing mice. Both bulk RNA sequencing (RNA-seq) and single-cell RNA-seq (scRNA-seq) were applied to portray the modulation of CKI combined with chemotherapy on the TME of TNBC mice. CKI significantly enhanced the anticancer activity of chemotherapy in vivo with no obvious side effects. Flow cytometry results revealed a significantly higher activation of CD8+ T lymphocytes in the spleens and tumors of the mice with combination therapy. Bulk RNA-seq indicated that CKI could promote the cytotoxic immune cell infiltrating into tumor tissues. Meanwhile, scRNA-seq further revealed that CKI combined with chemotherapy could enhance the percentage of tumor-infiltrating CD8+ T cells, inhibit tumor-promoting signaling pathways, and promote T cell activation and positive regulation of immune response. In addition, CKI showed obvious anticancer activity against MDA-MB-231 breast tumor cells in vitro. The combination of CKI and chemotherapy might provide a higher efficiency and lower toxicity strategy than a single chemotherapy drug for TNBC. CKI potentiates the anti-TNBC effects of chemotherapy by activating anti-tumor immune response in mice.

Gold Nanoparticle (AuNP) as a Therapeutic Enhancer for Radio – And Immunotherapy Therapy Combination in Triple Negative Breast Cancer

<h3>Purpose/Objective(s)</h3> Triple negative breast cancer (TNBC) is the most aggressive breast cancer (BC) form, with a high metastases rate and a very low survival. The aggressiveness of TNBC coupled with a significant toxicity and suboptimal chemotherapy outcomes underscores the urgency for new TNBC treatments. In recent years, immunotherapy has emerged as a promising option. In particular, immune checkpoint blockers (ICB) targeting PD-L1/PD1 inhibitory T cell check point pathway showed clinical responses and have been explored for TNBC. Unfortunately, the response rates to standalone ICB therapy are low (15-20%), indicating the presence of inhibitory immune mechanisms. Radiation therapy (RT) has been widely used in BC therapies. In addition to antitumor (antiproliferative) effects, RT has been evidenced to stimulate immune tumor rejection through immunomodulation of the tumor microenvironment (TME) that has been shown to enhance the response to immunotherapy in mouse BC models. Antitumor RT effects, including TME immunomodulation, can be improved by using radiosensitizers, such as gold nanoparticles (AuNPs). We hypothesize that AuNP potentiates RT-induced immunomodulatory effects, leading to a more efficient response to ICB in TNBC. To test this hypothesis, we used AuNP as an enhancer of RT-induced immunological TME changes, to improve ICB therapy response in murine orthotopic syngeneic 4T1Luc TNBC model. <h3>Materials/Methods</h3> Female Balb/c mice bearing 4T1Luc tumors received intratumoral injections of 14 nm AuNPs. After 24h mice were irradiated with fractionated regimen of 3 × 6 Gy dose using 225 kV photons. After the 3rd RT dose, mice received 3 doses of anti-PD-L1 antibody that were 4 days apart. Therapeutic efficiency was determined by assessing the tumor growth and animal survival. Tumor tissue immunohistochemistry determined the expression of TME immunological markers and immune cell tumor infiltration. <h3>Results</h3> AuNPs improved response to anti PD-L1 treatment in mice receiving RT, shown by significant delay in tumor growth and increase in survival compared to the animals receiving RT+ AuNP (p<0.01) and to the animals receiving RT+ anti PD-L1 or RT alone (p<0.05). These results were accompanied with changes in the expression of TME immunological markers and T cell and macrophage infiltration. <h3>Conclusion</h3> In TNBC patients, induction of antitumor immune response may play a critical role in improving clinical outcomes. Here we show that AuNP enhanced the effect of a fractionated RT regimen that has significantly improved the response to anti PD-L1 treatment in 4T1Luc TNBC mouse model. This effect was measured by a delay in tumor growth and an increase in animal survival. These findings support the role of immunological mechanisms in TNBC and provide a platform for designing multimodal TNBC RT formulations with novel radiosensitizers or immunotherapy.

OSW-1 induces apoptosis and cyto-protective autophagy, and synergizes with chemotherapy on triple negative breast cancer metastasis.

Triple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer. As yet, chemotherapy with drugs such as doxorubicin is the main treatment strategy. However, drug resistance and dose-dependent toxicities restrict their clinical use. Natural products are major sources of anti-tumor drugs. OSW-1 is a natural compound with strong anti-cancer effects in several types of cancer, but its effects on the efficacy of chemotherapy in TNBC and its underlying mechanism remain unclear. The inhibitory activities of OSW-1 and its combination with several chemotherapy drugs were tested using in vitro assays and in vivo subcutaneous and metastatic mouse TNBC models. The effects of the mono- and combination treatments on TNBC cell viability, apoptosis, autophagy and related signaling pathways were assessed using MTT, flow cytometry, RNA sequencing and immunology-based assays. In addition, the in vivo inhibitory effects of OSW-1 and (combined) chemotherapies were evaluated in subcutaneous and metastatic mouse tumor models. We found that OSW-1 induces Ca2+-dependent mitochondria-dependent intrinsic apoptosis and cyto-protective autophagy through the PI3K-Akt-mTOR pathway in TNBC cells in vitro. We also found that OSW-1 and doxorubicin exhibited strong synergistic anti-TNBC capabilities both in vivo and in vitro. Combination treatment strongly inhibited spontaneous and experimental lung metastases in 4T1 mouse models. In addition, the combination strategy of OSW-1 + Carboplatin + Docetaxel showed an excellent anti-metastatic effect in vivo. Our data revealed the mode of action and molecular mechanism underlying the effect of OSW-1 against TNBC, and provided a useful guidance for improving the sensitivity of TNBC cells to conventional chemotherapeutic drugs, which warrants further investigation.

TGFB2-AS1 inhibits triple-negative breast cancer progression via interaction with SMARCA4 and regulating its targets TGFB2 and SOX2

Triple-negative breast cancer (TNBC) is the most challenging breast cancer subtype for its high rates of relapse, great metastatic potential, and short overall survival. How cancer cells acquire metastatic potency through the conversion of noncancer stem-like cells into cancer cells with stem-cell properties is poorly understood. Here, we identified the long noncoding RNA (lncRNA) TGFB2-AS1 as an important regulator of the reversibility and plasticity of noncancer stem cell populations in TNBC. We revealed that TGFB2-AS1 impairs the breast cancer stem-like cell (BCSC) traits of TNBC cells invitro and dramatically decreases tumorigenic frequency and lung metastasis invivo. Mechanistically, TGFB2-AS1 interacts with SMARCA4, a core subunit of the SWI/SNF chromatin remodeling complex, and results in transcriptional repression of its target genes including TGFB2 and SOX2 in an in cis or in trans way, leading to inhibition of transforming growth factor β (TGFβ) signaling and BCSC characteristics. In line with this, TGFB2-AS1 overexpression in an orthotopic TNBC mouse model remarkably abrogates the enhancement of tumor growth and lung metastasis endowed by TGFβ2. Furthermore, combined prognosis analysis of TGFB2-AS1 and TGFβ2 in TNBC patients shows that high TGFB2-AS1 and low TGFβ2 levels are correlated with better outcome. These findings demonstrate a key role of TGFB2-AS1 in inhibiting disease progression of TNBC based on switching the cancer cell fate of TNBC and also shed light on the treatment of TNBC patients.

Oligo-Fucoidan supplementation enhances the effect of Olaparib on preventing metastasis and recurrence of triple-negative breast cancer in mice

BackgroundSeaweed polysaccharides have been recommended as anticancer supplements and for boosting human health; however, their benefits in the treatment of triple-negative breast cancers (TNBCs) and improving immune surveillance remain unclear. Olaparib is a first-in-class poly (ADP-ribose) polymerase inhibitor. Oligo-Fucoidan, a low-molecular-weight sulfated polysaccharide purified from brown seaweed (Laminaria japonica), exhibits significant bioactivities that may aid in disease management.MethodsMacrophage polarity, clonogenic assays, cancer stemness properties, cancer cell trajectory, glucose metabolism, the TNBC 4T1 cells and a 4T1 syngeneic mouse model were used to inspect the therapeutic effects of olaparib and Oligo-Fucoidan supplementation on TNBC aggressiveness and microenvironment.ResultsOlaparib treatment increased sub-G1 cell death and G2/M arrest in TNBC cells, and these effects were enhanced when Oligo-Fucoidan was added to treat the TNBC cells. The levels of Rad51 and programmed death-ligand 1 (PD-L1) and the activation of epidermal growth factor receptor (EGFR) and adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) facilitate drug resistance and TNBC metastasis. However, the combination of olaparib and Oligo-Fucoidan synergistically reduced Rad51 and PD-L1 levels, as well as the activity of EGFR and AMPK; consistently, TNBC cytotoxicity and stemness were inhibited. Oligo-Fucoidan plus olaparib better inhibited the formation of TNBC stem cell mammospheroids with decreased subpopulations of CD44high/CD24low and EpCAMhigh cells than monotherapy. Importantly, Oligo-Fucoidan plus olaparib repressed the oncogenic interleukin-6 (IL-6)/p-EGFR/PD-L1 pathway, glucose uptake and lactate production. Oligo-Fucoidan induced immunoactive and antitumoral M1 macrophages and attenuated the side effects of olaparib, such as the promotion on immunosuppressive and protumoral M2 macrophages. Furthermore, olaparib plus Oligo-Fucoidan dramatically suppressed M2 macrophage invasiveness and repolarized M2 to the M0-like (F4/80high) and M1-like (CD80high and CD86high) phenotypes. In addition, olaparib- and Oligo-Fucoidan-pretreated TNBC cells resulted in the polarization of M0 macrophages into CD80(+) M1 but not CD163(+) M2 macrophages. Importantly, olaparib supplemented with oral administration of Oligo-Fucoidan in mice inhibited postsurgical TNBC recurrence and metastasis with increased cytotoxic T cells in the lymphatic system and decreased regulatory T cells and M2 macrophages in tumors.ConclusionOlaparib supplemented with natural compound Oligo-Fucoidan is a novel therapeutic strategy for reprogramming cancer stemness, metabolism and the microenvironment to prevent local postsurgical recurrence and distant metastasis. The combination therapy may advance therapeutic efficacy that prevent metastasis, chemoresistance and mortality in TNBC patients.

Tumor Microenvironment Multiple Responsive Nanoparticles for Targeted Delivery of Doxorubicin and CpG Against Triple-Negative Breast Cancer.

IntroductionCurrently, the main treatment for advanced breast cancer is still chemotherapy. Immunological and chemical combination therapy has a coordinated therapeutic effect and achieves some efficacy. However, the immunosuppressive tumor microenvironment is a major cause for the failure of immunotherapy in breast cancer. CpG oligodeoxynucleotides can activate the tumor immune microenvironment to reverse the failure of immunotherapy.MethodsIn this study, we designed an amphiphilic peptide micelle system (Co-LMs), which can targeted delivery of the immune adjuvant CpG and the chemotherapeutic drug doxorubicin to breast cancer tumors simultaneously. The peptide micelle system achieved tumor microenvironment pH and redox-sensitive drug release.Results and DiscussionCo-LMs showed 2.3 times the antitumor efficacy of chemotherapy alone and 5.1 times the antitumor efficacy of immunotherapy alone in triple-negative breast cancer mice. Co-LMs activated cytotoxic CD8+ T lymphocytes and CD4+ T cells in mice to a greater extent than single treatments. We also found that Co-LMs inhibited the metastasis of circulating tumor cells in the bloodstream to some extent. These results indicate that the Co-LMs offer a promising therapeutic strategy against triple-negative breast cancer.

Albumin-Coated Framework Nucleic Acids as Bionic Delivery System for Triple-Negative Breast Cancer Therapy

Triple-negative breast cancer (TNBC) is a subtype of breast cancer, and it has aggressive and more frequent tissue metastases than other breast cancer subtypes. Because the proliferation of TNBC tumor cells does not depend on estrogen receptor (ER), progesterone receptor (PR), and Erb-B2 receptor tyrosine kinase 2 (HER2) and lacks accurate drug targets, conventional chemotherapy is challenging to be effective, and adverse reactions are severe. At present, the treatment strategy for TNBC generally depends on a combination of surgery, radiotherapy, and chemotherapy. Conventional administration methods have minimal effects on TNBC and cause severe damage to normal tissues. Therefore, it is an urgent task to develop an efficient and practical way of drug delivery and open up a new horizon of targeted therapy for TNBC. In our work, bovine serum albumin (BSA) acted as the protective film to prolong the circulation time of the tetrahedral framework nucleic acid (tFNA) delivery system and resist immune clearance in vivo. tFNA was used as a carrier loaded with DOX and AS1411 aptamers for the targeted treatment of triple-negative breast cancer. Compared with existing approaches, this optimized system exhibits stronger tumor-targeting so that tFNAs can be more concentrated around the tumor tissue, reducing DOX toxicity to other organs. This bionic delivery system exhibited effective tumor growth inhibition in the TNBC mice model, offering the clinical potential to promote the treatment of TNBC with great potential for clinical translation.

Inhibition of Mitochondrial Biosynthesis Using a "Right-Side-Out" Membrane-Camouflaged Micelle to Facilitate the Therapeutic Effects of Shikonin on Triple-Negative Breast Cancer.

The mitochondria represent a potential target for the treatment of triple-negative breast cancer (TNBC) and shikonin (SK) has shown remarkable therapeutic effects on TNBC. Herein, it is found that SK possesses potent inhibitory effects on mitochondrial biogenesis via targeting polymerase gamma (POLG). However, its application is restricted by its poor aqueous solubility and stability, and therefore, a biomimetic micelle to aid with tumor lesion accumulation and mitochondria-targeted delivery of SK is designed. A folic acid (FA) conjugated polyethylene glycol derivative (FA-PEG-FA) is inserted onto the external membranes of red blood cells (FP-RBCm) to prepare a "right-side-out" RBCm-camouflaged cationic micelle (ThTM/SK@FP-RBCm). Both FP-RBCm coating and a triphenylphosphine (TPP) moiety on the periphery of micelles contribute to tumor lesion distribution, receptor-mediated cellular uptake, and electrostatic attraction-dependent mitochondrial targeting, thereby maximizing inhibitory effects on mitochondrial biosynthesis in TNBC cells. Intravenous administration of ThTM/SK@FP-RBCm leads to profound inhibition of tumor growth and lung metastasis in a TNBC mouse model with no obvious toxicity. This work highlights the mitochondria-targeted delivery of SK using a "right-side-out" membrane-camouflaged micelle for the inhibition of mitochondrial biogenesis and enhanced therapeutic effects on TNBC.

Radiation enhances the efficacy of EGFR-targeted CAR-T cells against triple-negative breast cancer by activating NF-κB/Icam1 signaling

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, with limited treatment options. Epidermal growth factor receptor (EGFR) is reported to be expressed in 50%-75% of TNBC patients, making it a promising target for cancer treatment. Here we show that EGFR-targeted chimeric antigen receptor (CAR) Tcell therapy combined with radiotherapy provides enhanced antitumor efficacy in immunocompetent and immunodeficient orthotopic TNBC mice. Intriguingly, this combination therapy resulted in a substantial increase in the number of tumor-infiltrating CAR-T cells. The efficacy of this combination was independent of tumor radiosensitivity and lymphodepleting preconditioning. Cytokine profiling showed that this combination did not increase the risk of cytokine release syndrome (CRS). RNA sequencing (RNA-seq) analysis revealed that EGFR-targeting CAR-T therapy combined with radiotherapy increased the infiltration of CD8+ T and natural killer (NK) cells into tumors. Mechanistically, radiation significantly increased Icam1 expression on TNBC cells via activating nuclear factor κB (NF-κB) signaling, thereby promoting CAR-T cell infiltration and killing. These results suggest that CAR-T therapy combined with radiotherapy may be a promising strategy for TNBC treatment.

Response to Treatment in 4T1 Tumor Following Exposure to Paclitaxel and Doxorubicin Based on Antiangiogenic Effects

Background: 4T1 is a mice transplantable mammary carcinoma cell line with highly tumorigenic and invasive properties, making it a suitable preclinical oncology model for triple-negative breast cancer (TNBC). This pilot study aimed to create a model of clinical stages in TNBC mice and to evaluate the response to treatment with paclitaxel (PTX) and doxorubicin (DOX) based on antiangiogenic effects.&#x0D; Methods: Syngeneic tumors were developed in BALB/c female mice by 4T1 cell line. The mice were randomly distributed into three different groups, each containing four. A group of four was considered as healthy normal. When tumor growth reached 100- 200 mm3 , two groups received the maximum tolerated dose (MTD) of PTX and DOX, respectively. Normal saline was injected into the sham control group. The tumors and tissue margins were removed by surgery one week following chemotherapy. Angiogenesis genes and microvessel density (MVD) were analyzed by real-time PCR and immunohistochemistry, respectively. Response to treatment was also assessed by standard methods of H&amp;E staining.&#x0D; Results: TNBC tumors were confirmed by pathological staining. The volume of tumors and the angiogenesis gene expressions of VEGFR1, VEGFR2, and HIF1α decreased in treated tumors compared to control (p &lt; 0.05). Response to treatment to PTX was more than DOX, and the MVD decreased in both PTX and DOX chemotherapy groups.&#x0D; Conclusion: Although PTX is more effective than DOX in reducing angiogenesis genes, both have the potential for treatment in the 4T1 mouse model.

Development of BET Inhibitors as Potential Treatments for Cancer: Optimization of Pharmacokinetic Properties.

We describe the synthesis of triazole-containing carboline derivatives and their utility as bromodomain and extra-terminal (BET) inhibitors. A convergent synthetic route permitted the detailed investigation of deuteration and fluorination strategies to reduce clearance while maintaining a favorable in vitro profile. This work led to the identification of a potent BET inhibitor, 2-{8-fluoro-3-[4-(2H3)methyl-1-methyl-1H-1,2,3-triazol-5-yl]-5-[(S)-(oxan-4-yl)(phenyl)methyl]-5H-pyrido[3,2-b]indol-7-yl}propan-2-ol (15), which demonstrated reduced clearance and an improved pharmacokinetic (PK) profile across preclinical species. Importantly, no major metabolite was observed when 15 was incubated with human hepatocytes (hHEP) for 2 h. This study culminated with the evaluation of 15 in a mouse triple-negative breast cancer (TNBC) tumor model where it demonstrated robust efficacy at low doses.

Olaparib@human serum albumin nanoparticles as sustained drug-releasing tumour-targeting nanomedicine to inhibit growth and metastasis in the mouse model of triple-negative breast cancer

Poly(ADP-ribose) polymerase inhibitor olaparib demonstrated therapeutic effectiveness in highly metastatic triple-negative breast cancer (TNBC). However, olaparib offers a weak therapeutic response in wild-type BRCA cancers due to the drug’s poor bioavailability. Here, a bioinspired/active-tumour targeted nanoparticles system of human serum albumin with physical entrapment of olaparib was prepared via a low-energy desolvation technique using the crosslinker glutaraldehyde. The developed OLA@HSA NPs were nanosize (∼140 nm), kinetically stable with a low polydispersity (0.3), exhibited olaparib entrapment (EE 76.01 ± 2.53%, DL 6.76 ± 0.22%) and sustained drug release at pH 7.4 with an enhancement of drug release in acidic pH. OLA@HSA NPs decreased the half-maximal inhibitory concentrations (IC50) of olaparib by 1.6-, 1.8-fold in 24 h and 2.2-, 2.4-fold in 48 h for human (MDA-MB 231) and mouse (4T1) TNBC cells, respectively, mediated by their enhanced time-dependent cellular uptake than free olaparib. The OLA@HSA NPs induced concentration-dependent phosphatidylserine (apoptotic marker) externalisation and arrested the cell population in the G2/M phase in both the tested cell lines at a higher level than free olaparib. The NPs formulation increased DNA fragmentation, mitochondrial membrane depolarisation and ROS generation than the free olaparib. The in vivo study conducted using 4T1-Luc tumour-bearing mice demonstrated strong tumour growth inhibitory potential of OLA@HSA NPs by elevating apoptosis ROS generation and reducing the level of the antiproliferative marker, Ki-67. OLA@HSA NPs reduced the occurrence of lung metastasis (formation of metastasis nodules decreased by ∼10-fold). OLA@HSA NPs could be a promising nanomedicine for the TNBC treatment.

Anisomycin inhibits angiogenesis, growth, and survival of triple-negative breast cancer through mitochondrial dysfunction, AMPK activation, and mTOR inhibition.

Aberrant upregulation of mitochondrial biogenesis is observed in breast cancer and holds potential therapeutic option. In our work, we showed that inhibition of mitochondrial function by anisomycin is effective against triple-negative breast cancer (TNBC). Anisomycin inhibits growth and induces caspase-dependent apoptosis in a panel of TNBC cell lines. Of note, anisomycin at a tolerable dose remarkably suppresses growth of TNBC in mice. In addition, anisomycin effectively targets breast cancer angiogenesis through inhibiting capillary network formation, migration, proliferation, and survival. Mechanistic studies show that although anisomycin activates p38 and JNK, their activations are not required for anisomycin's action. In contrast, anisomycin inhibits mitochondrial respiration, and decreases mitochondrial membrane potential and adenosine triphosphate (ATP) level. The inhibitory effect of anisomycin is significantly reversed in mitochondria respiration-deficient ρ0 cells. As a consequence, anisomycin activates AMPK and inhibits mammalian target-of-rapamycin signaling pathways. Our work demonstrated that anisomycin is a useful addition to the treatment armamentarium for TNBC.

Abstract 3295: The Nanoprimer: A nanoparticle designed to the efficacy of nucleic acid-based therapeutics in oncology

Abstract Nucleic acid drugs have shown promise in oncology, however their use is limited by hepatic clearance. Due to the mononuclear phagocytic system (MPS), a large part of the intravenously-administered dose does not reach the therapeutic target. Our approach aims to address this issue using biocompatible nanoparticles—the Nanoprimer—that is administered prior to the therapeutic. Due to its physico-chemical properties, the Nanoprimer is transiently internalized by Kupffer cells (KC), and endothelial cells of the MPS, enabling temporary reduction of hepatic clearance. These data further explore the Nanoprimer’s mechanism of action and demonstrate that scavenger receptor-A (SR) is the main receptor involved in Nanoprimer binding and uptake by KC. Efficacy studies in mouse model show that the Nanoprimer dramatically increases the anti-tumor efficacy of nucleic acid-based treatment without introducing additional toxicities. An in vitro toxicity evaluation was performed investigating the Nanoprimer’s hemolytic properties and its potential ability to activate the complement system, platelet aggregation and pro-inflammatory cytokines*. SR-mediated binding and uptake of Nanoprimer by KC were assessed using a competitive binding assay with SR inhibitor polyinosinic acid. The impact of the Nanoprimer on anti-tumor efficacy was evaluated on orthotopic triple negative breast cancer (TNBC) model in combination with siRNA-loaded lipid nanoparticles (LNPs) targeting a novel cancer-specific gene transcript of a master regulator of invadopodia formation and Wnt, Hedgehog and Notch oncogenic pathways. Results showed that the Nanoprimer is safe at the effective dose. The Nanoprimer does not activate the complement components, induce hemolysis, platelet aggregation, or cytokine storm syndrome elements. The competitive inhibition of SR-A decreases Nanoprimer binding and KC-uptake by 70%, indicating SR-A selective uptake of the Nanoprimer by macrophages. The efficacy study shows that combining the Nanoprimer with siRNA LNP therapy significantly increased treatment efficiency, with a mean tumor growth inhibition ratio of 36.3% for LNPs alone versus 84.6% for the combination. Moreover, the combination therapy more efficiently prevents the development of pulmonary metastatic tumors, with a metastasis-inhibition rate of 98%. These data demonstrate the safety of the Nanoprimer, along with its ability to maximize the inhibition of primary tumor growth and pulmonary metastasis by systemic siRNA LNP therapy in a TNBC mouse model. They also highlight the capacity of Nanoprimer technology to unlock the potential of nucleic acid-based treatments. The ubiquitous nature of its mode of action allows its application to a broad spectrum of anti-cancer therapeutics. *we acknowledge NCL, Frederick National Laboratory for Cancer Research sponsored by the NCI for the help with conducting these experiments Citation Format: Julie Devallière, Laurence Poul, Maxime Bergère, Audrey Darmon, Océane Jibault, Francis Mpambani, Kelvin Tsai, Matthieu Germain. The Nanoprimer: A nanoparticle designed to the efficacy of nucleic acid-based therapeutics in oncology [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 3295.

Abstract 3519: Bioengineering enucleated cell vehicles for targeted delivery of Interleukin 12 to metastatic tumors

Abstract Metastatic triple negative breast cancer (TNBC) includes poor prognoses and limited effective therapies. Metastasis is the major barrier to durable clinical responses as many patients appear cured of their disease, yet return to clinic with recurrences from dormant micro-metastases. Immune activating agents, such as Interleukin 12 (IL-12) have attracted attention as anti-cancer adjuvants due to their ability to elicit systemic anti-cancer immunity. Although targeting distributed metastases can be accomplished by intravenous (IV) administration, administration of IL-12 into patient vasculature is highly inefficient due to its short half-life and poor toxicity profile. There is a major unmet need to develop biocompatible carriers that improve targeted delivery of IL-12 by specifically homing to metastatic lesions. Mesenchymal stem cells (MSCs) are considered ideal biodelivery vehicles as they display innate tumor-trophism and can deliver biomolecules specifically to systemic metastases. However, major issues need to be addressed before MSCs can be used to deliver IL-12 to patients: MSC tumor homing needs to be improved, and unmodified MSCs can engraft and contribute to tumor growth and metastasis, raising legitimate safety concerns. To address these major limitations, our laboratory developed a MSC-derived biodelivery vehicle with enhanced homing abilities to deliver biomolecules directly to tumors. Our approach involved using scRNA-seq to identify chemoattractant receptor/ligand pairs (CXCR4/CXCL12, CCR2/CCL2) and endothelial adhesion molecules (PSGL-1/P-E-Selectins) that mediate leukocyte homing and extravasation. We then lentivirally expressed these receptors in MSCs and removed nuclei using Ficoll gradient ultracentrifugation. These bioengineered enucleated MSCs (termed Cargocytes™) have a defined lifespan (4 days) and robustly home to TNBC. This is an important breakthrough as TNBC-trophic Cargocytes can be scaled for clinical applications and are safe for in vivo use as described in our recent publication (Nature Biomedical Engineering, in press). By using syngeneic mouse models of metastatic TNBC, we have observed IV-injected Cargocytes robustly extravasate and precisely home to metastatic lung nodules, suggesting Cargocytes act as remarkable biodelivery vehicles. Indeed, the tumor-trophism of Cargocytes includes both integrating into large macro-metastases and homing to broadly distributed micro-metastases. Cargocytes effectively produce and deliver high doses of IL-12 to mouse TNBC, which induces strong immune activation in tumors. This immune activation involved a potent anti-metastatic response, evidencing strong therapeutic efficacy. These findings suggest Cargocyte-delivered IL-12 could constitute a safe, potent, and highly specific anti-metastatic therapy for TNBC and other metastatic cancers sharing similar chemoattractant profiles. Citation Format: Jeffrey White, Huawei Wang, Flavia Franco Da Cunha, Maureen Ruchhoeft, Richard Klemke. Bioengineering enucleated cell vehicles for targeted delivery of Interleukin 12 to metastatic tumors [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 3519.

Abstract 5399: Synergistic combination nanomedicine of doxorubicin and oligo hyaluronic acid inhibits DNA damage repair and overcomes drug resistance in metastatic triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype. Despite the early response to chemotherapy, high incidence of recurrence leads to short overall survival and poor prognosis. Native hyaluronic acid (HA) interacts with CD44 and receptors for hyaluronan mediated motility (RHAMM) overexpressed in TNBC mediating chemo-resistance and metastasis. In contrast, oligomeric HA (oHA) can disrupt HA-CD44/RHAMM interactions and attenuate oncogenic and pro-metastatic pathways, such as the mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) pathway, reducing downstream DNA repair and drug resistance markers. Our previous work demonstrated that oHA can exert synergistic anti-tumor and anti-metastatic effects when combined with doxorubicin (DOX) and co-loaded in an integrin-targeted iRGD-modified nanoparticle system (iRGD-DOX-oHA-PLNs). This study aims to investigate the inhibitory effect of iRGD-DOX-oHA-PLNs on both DNA single-strand break (SSB) and double-strand break (DSB) repair proteins and drug efflux pumps responsible for multidrug resistance to enhance DOX efficacy in breast cancer gene 1 (BRCA1) mutant and non-mutant TNBC. Methods: The cytotoxicity of DOX, oHA and their combinations in free solution or in nanoparticles was evaluated by clonogenic assay in human MDA-MB-231-luc-D3H2LN and MDA-MB-436 (BRCA1 mutant) TNBC cells. The in vitro expression of a DNA DSB marker, DNA repair markers, and drug efflux pump P-glycoprotein (P-gp) were measured by Western blot. The in vivo expression level of BRCA1 and Rad51 in an orthotopic TNBC mouse model was determined by immunohistochemical staining. Results: The combination of DOX-oHA showed synergism against both BRCA1 mutant and non-mutant TNBC cells. The iRGD-DOX-oHA-PLNs induced great increases in DNA DSBs demonstrated by the highest γH2AX level compared to other treatment groups. These nanoparticles also showed inhibitory effects on the expression of both DNA SSB repair protein (poly (ADP-ribose) polymerase) and DNA DSB repair proteins (Rad50 and Rad51), contributing to the enhanced efficacy of chemotherapy. The immunohistochemical staining of tumor tissues indicated lower levels of BRCA1 and Rad51 after iRGD-DOX-oHA-PLN treatment than the formulation without oHA, attributable to the effect of intracellularly delivered oHA on limiting the MAPK signaling. Additionally, iRGD-DOX-oHA-PLNs reduced the expression of the drug efflux pump P-gp as compared to DOX treatment groups without oHA. Conclusion: The co-delivery of oHA and DOX in the iRGD-DOX-oHA-PLNs efficiently blocked DNA damage repair and down-regulated the drug efflux pump P-gp, thus improving the efficacy of DOX. Collectively, this nanoparticle system could be a promising option for metastatic TNBC treatment. Citation Format: Pei Zhi, Ibrahim Alradwan, Tian Zhang, HoYin Lip, Abdulmutalib Zetrini, Chunsheng He, Jeffery Henderson, Andrew Michael Rauth, Xiao Yu Wu. Synergistic combination nanomedicine of doxorubicin and oligo hyaluronic acid inhibits DNA damage repair and overcomes drug resistance in metastatic triple-negative 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 5399.

Abstract 5304: A promising combination partner for paclitaxel in triple-negative breast cancer (TNBC) therapy

Abstract Cytotoxic chemotherapy is initially effective in patients with metastatic triple-negative breast cancer (mTNBC). However, most of the TNBC patients have high risk of recurrence after chemotherapy and metastatic tumor recurrence indicates rapid tumor proliferation and drug resistance. In TNBC cells, treatment with the chemotherapeutic drug paclitaxel (PTX) induces Transforming growth factor (TGF)-β signaling pathway. TGF-β is a master regulator of tumor angiogenesis and epithelial-mesenchymal transition (EMT), which are limiting the efficacy of cancer therapies and suppressing the anti-tumor immune response. TGF-β induces autophagy in tumor cells and autophagy is closely associated with cancer cell survival and contributing to resistance to therapies. The targeting of TGF-β pathway is possibly an effective strategy to inhibit drug resistance and increase the sensitivity of cancer treatment. Charis 1000 (C1K), which is currently under development, is a synthetic peptide that targets TGF-β1. As C1K partially inhibits TGF-β1-induced Smad2 phosphorylation in TNBC cells, there is no risk of side effects unlike small molecules or antibodies that completely inhibit the TGF-β1 signaling pathway. Combination therapies including chemotherapy are actively investigated as effective treatments for various cancers. PTX, one of frequently used drugs in combination therapies, increases phosphorylation of Smad2, a TGF- β1 downstream signaling pathway and induces autophagy in TNBC cells, but PTX in combination with C1K increases apoptosis while reducing Smad2 phosphorylation and autophagy. The combination of C1K with PTX in the mouse TNBC model showed significant tumor size reduction in comparison to the PTX alone group. In addition, the combination of C1K with PTX confirmed the increased apoptosis and decreased autophagy in the tumor tissues. Furthermore, the combination of C1K with PTX increased the survival rate of mice that was reduced due to the side effects of PTX. These data indicate that the drug resistance induced by TGF-β signaling pathway can be suppressed by C1K combination through TGF-β signaling modulation. C1K may be a promising combination partner for cancer treatment by reducing drug resistance and increasing apoptotic potential of anticancer drugs. Currently, a phase1 clinical trial of the C1K combination therapy with PTX is ongoing and the preclinical studies of the C1K combination with immune checkpoint inhibitors (ICIs) are actively investigated as well. Citation Format: Youl-Nam Lee, Yun-hee Han, Won-Sam Kim, Cheol-Min Lee, Eun-Joung Moon, Hae-Jin Kim. A promising combination partner for paclitaxel in triple-negative breast cancer (TNBC) therapy [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 5304.

Abstract 5476: Novel azetidine-based STAT3 inhibitors induce misfolded protein response, endoplasmic reticulum stress and mitophagy, and inhibit breast tumor growth in vivo

Abstract Anticancer drugs that directly target Signal Transducer and Activator of Transcription (Stat)3 have remained elusive. We present azetidine-based small molecules as a new class of potent Stat3 inhibitors. Compounds H172 and H182 have potency (IC50) of 0.38-0.98 µM, H105 and H120 have IC50 of 1.75-2.07 µM against Stat3 DNA-binding activity in vitro, compared to the IC50 of &amp;gt;17 µM against Stat1 or Stat5 activity. Treatment of the triple-negative breast cancer (TNBC), MDA-MB-231 and MDA-MB-468 cells with the new compounds inhibited constitutive Stat3 activation, promoted Stat3 ubiquitination, blocked anchorage-dependent and independent growth, with potency, EC50 of 1.0-1.9 μM, and induced apoptosis of these cells. By contrast, pYStat1, pYStat5, pS727Stat3, pSrc, pJanus kinase, or the induction of other signaling events are not inhibited in compound-treated cells, and normal or tumor cells that do not harbor aberrantly-active Stat3 are only weakly inhibited. In vivo delivery of H120 or H182 as a single agent inhibited growth of human TNBC xenografts, and of H278 (HCl salt of H182) combined with radiation abrogated tumor growth and improved survival in mouse TNBC syngeneic models. These studies provide novel mechanism of disruption of aberrantly-active Stat3 and its dynamics in showing that the azetidine-based Stat3 inhibitors trigger Stat3 ubiquitination, and tumor cell death. H182 and H172 are new entities for further therapeutic development against TNBC and other cancers that harbor aberrantly-active Stat3. Citation Format: Peibin Yue, Francisco Lopez-Tapia, Yinsong Zhu, Nagendra Verma, Felix Alonso-Valenteen, Simoun Mikhael, Lali Medina-Kauwe, Marcus Tius, James Turkson. Novel azetidine-based STAT3 inhibitors induce misfolded protein response, endoplasmic reticulum stress and mitophagy, and inhibit breast tumor growth in vivo [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 5476.