Triple-negative Breast Cancer Models Research Articles

Preclinical evaluation of the theranostic potential of 89Zr/177Lu-labeled anti-TROP-2 antibody in triple-negative breast cancer model

BackgroundTriple-negative breast cancer (TNBC) is one of the most lethal malignant tumors among women, characterized by high invasiveness, high heterogeneity, and lack of specific therapeutic targets such as estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Trophoblast cell-surface antigen-2 (TROP-2) is a transmembrane glycoprotein over-expressed in 80% of TNBC patients and is associated with the occurrence, progress, and poor prognosis of TNBC. The TROP-2 targeted immunoPET imaging allows non-invasive quantification of the TROP-2 expression levels of tumors, which could help to screen beneficiaries most likely to respond to SG and predict the response. This study aimed to develop a 89Zr/177Lu-radiolabeled anti-TROP-2 antibody (NY003) for immunoPET and SPECT imaging, as well as radioimmunotherapy (RIT) in TROP-2 (+)TNBC tumor-bearing model. Based on the camelid antibody, we developed a TROP-2 targeted recombinant antibody NY003. NY003 was conjugated with DFO and DTPA for 89Zr and 177Lu radiolabelling, respectively. The theranostic potential of [89Zr]Zr-DFO-NY003/[177Lu]Lu-DTPA-NY003 was evaluated through immunoPET, SPECT imaging, and RIT studies in the subcutaneous TROP-2 positive TNBC xenograft mice model.ResultsThe high binding affinity of NY003 to TROP-2 was verified through ELISA. The radiochemical purity of [89Zr]Zr-DFO-NY003/[177Lu]Lu-DTPA-NY003 exceeded 95% and remained stable within 144h p.i. in vitro. ImmunoPET and SPECT imaging showed the specific accumulation of [89Zr]Zr-DFO-NY003/[177Lu]Lu-DTPA-NY003 in MDA-MB-231 tumors and gradually increased with the time tested, significantly higher than that in control groups (P < 0.05). The strongest anti-tumor efficacy was observed in the high-dose of [177Lu]Lu-DTPA-NY003 group, followed by the low-dose group, the tumor growth was significantly suppressed by [177Lu]Lu-DTPA-NY003, the tumor volumes of both high- and low-dose groups were smaller than the control groups (P < 0.05). Ex vivo biodistribution and histological staining verified the results of in vivo imaging and RIT studies.ConclusionAs a drug platform for radiotheranostics, 89Zr/177Lu-radiolabeled anti-TROP-2 antibody NY003 could not only non-invasively screen the potential beneficiaries for optimizing SG ADC treatment but also suppressed the growth of TROP-2 positive TNBC tumors, strongly supporting the theranostic potential of [89Zr]Zr-DFO-NY003/[177Lu]Lu-DTPA-NY003.

Digoxin-Mediated Inhibition of Potential Hypoxia-Related Angiogenic Repair in Modulated Electro-Hyperthermia (mEHT)-Treated Murine Triple-Negative Breast Cancer Model.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer type with no targeted therapy and hence limited treatment options. Modulated electrohyperthermia (mEHT) is a novel complementary therapy where a 13.56 MHz radiofrequency current targets cancer cells selectively, inducing tumor damage by thermal and electromagnetic effects. We observed severe vascular damage in mEHT-treated tumors and investigated the potential synergism between mEHT and inhibition of tumor vasculature recovery in our TNBC mouse model. 4T1/4T07 isografts were orthotopically inoculated and treated three to five times with mEHT. mEHT induced vascular damage 4-12 h after treatment, leading to tissue hypoxia detected at 24 h. Hypoxia in treated tumors induced an angiogenic recovery 24 h after the last treatment. Administration of the cardiac glycoside digoxin with the potential hypoxia-inducible factor 1-α (HIF1-α) and angiogenesis inhibitory effects could synergistically augment mEHT-mediated tumor damage and reduce tissue hypoxia signaling and consequent vascular recovery in mEHT-treated TNBC tumors. Conclusively, repeated mEHT induced vascular damage and hypoxic stress in TNBC that promoted vascular recovery. Inhibiting this hypoxic stress signaling enhanced the effectiveness of mEHT and may potentially enhance other forms of cancer treatment.

Characterization of immunomodulating agents from Staphylococcus aureus for priming immunotherapy in triple-negative breast cancers

Immunotherapy, specifically immune checkpoint blockade (ICB), has revolutionized the treatment paradigm of triple-negative breast cancers (TNBCs). However, a subset of TNBCs devoid of tumor-infiltrating T cells (TILs) or PD-L1 expression generally has a poor response to immunotherapy. In this study, we aimed to sensitize TNBCs to ICB by harnessing the immunomodulating potential of S. aureus, a breast-resident bacterium. We show that intratumoral injection of spent culture media from S. aureus recruits TILs and suppresses tumor growth in a preclinical TNBC model. We further demonstrate that α-hemolysin (HLA), an S. aureus-produced molecule, increases the levels of CD8+ T cells and PD-L1 expression in tumors, delays tumor growth, and triggers tumor necrosis. Mechanistically, while tumor cells treated with HLA display Gasdermin E (GSDME) cleavage and a cellular phenotype resembling pyroptosis, splenic T cells incubated with HLA lead to selective expansion of CD8+ T cells. Notably, intratumoral HLA injection prior to ICB augments the therapeutic efficacy compared to ICB alone. This study uncovers novel immunomodulatory properties of HLA and suggests that intratumoral administration of HLA could be a potential priming strategy to expand the population of TNBC patients who may respond to ICB.

Adoptive cell transfer therapy with ex vivo primed peripheral lymphocytes in combination with anti-PDL1 therapy effectively inhibits triple-negative breast cancer growth and metastasis

BackgroundAdoptive cell transfer cancer immunotherapy holds promise for treating disseminated disease, yet generating sufficient numbers of lymphocytes with anti-cancer activity against diverse specificities remains a major challenge. We recently developed a novel procedure (ALECSAT) for selecting, expanding and maturating polyclonal lymphocytes from peripheral blood with the capacity to target malignant cells.MethodsImmunodeficient mice were challenged with triple-negative breast cancer cell lines or patient-derived xenografts (PDX) and treated with allogeneic or autologous ALECSAT cells with and without anti-PDL1 therapy to assess the capacity of ALECSAT cells to inhibit primary tumor growth and metastasis.ResultsALECSAT mono therapy inhibited metastasis, but did not inhibit primary tumor growth or prolong survival of tumor-bearing mice. In contrast, combined ALECSAT and anti-PDL1 therapy significantly inhibited primary tumor growth, nearly completely blocked metastasis, and prolonged survival of tumor-bearing mice.ConclusionsCombined ALECSAT and anti-PDL1 therapy results in favorable anti-cancer responses in both cell line-derived xenograft and autologous PDX models of advanced triple-negative breast cancer.

Ginger volatile oil inhibits the growth of MDA-MB-231 in the bisphenol A environment by altering gut microbial diversity

ObjectiveTo examine the impact of ginger volatile oil (GVO) on the growth of MDA-MB-231 breast cancer cells in the presence of bisphenol A (BPA) by modulating the diversity of gut microbiota. Methods: MDA-MB-231 breast cancer cells were injected subcutaneously into the right armpit of female BALB/c Nude (nu/nu) mice to create a triple negative breast cancer model. Thirty nude mice were randomly divided into 5 groups: control group (distilled water every day), BPA control group (distilled PEG-400+ DMSO + cyclodextrin every day), BPA + GVO (0.25 mL/kg) group, BPA + GVO (0.5 mL/kg) group, BPA + GVO (1 mL/kg) group, 6 mice in each group; The drug was given by gavage once a day for 4 weeks. At the end of the experiment, the changes of tumor mass and tumor volume were observed and compared in 5 groups of tumor-bearing mice. High-throughput sequencing (16S rRNA) was used to detect the changes of gut microflora in each group. Results: The volume and weight of breast cancer decreased in the low, medium and high dose groups of GVO. Among them, the difference between the high-dose group and the BPA group reached a significant level (P < 0.05). The species and abundance of gut flora decreased following BPA treatment, but increased after combined treatment of BPA with GVO. In the tumor control group, the ratio of Firmicutes(F) and Bacteroidea(B) respectively was 0.10:0.79 at the phylum level, while the ratio of BPA group further decreased (0.04:0.88). After feeding GVO, the number of Firmicutes and Bacteroidea increased, the F/B ratio increased, and the level of Lactobacillus and alistipes increased. In the BPA and GVO treatment group, the predominant gut microflora functions are cell membrane biogenesis, carbohydrate transport and metabolism. This is followed by amino acid transport and metabolism, and transcription function. After GVO administration, the Gram-positive bacteria (G+) ratio had an increasing trend and the Gram-negative bacteria (G−)ratio had a decreasing trend. Conclusion: The species and abundance of gut flora decreased following BPA treatment, but increased after combined treatment of BPA with GVO.

Theranostic Phthalocyanine and Naphthalocyanine Nanoparticles for Photoacoustic Imaging and Photothermal Therapy of Tumors.

Background: Phthalocyanine (PC) and naphthalocyanine (NC) dyes have long garnered interest as theranostic agents for optical imaging and phototherapy due to their near-infrared absorbance, photostability, imaging contrast, and proven safety in clinical trials. Yet, only a small fraction of these dyes has been evaluated as photothermal therapy (PTT) agents for cancer treatment. Methods: Nearly 40 distinct NC and PC dyes were encapsulated within polymeric PEG-PCL micelles via oil-in-water emulsions. The optimal NC/PC-loaded micelle formulations for PTT and photoacoustic (PA) imaging were identified through in vivo and in vitro studies. Results: The most promising candidate, CuNC(Octa)-loaded micelles, demonstrated a strong PA signal with a peak absorbance at ~870 nm, high photothermal efficiency, and photostability. The CuNC(Octa)-loaded micelles exhibited heat generation as good or better than gold nanorods/nanoshells and >10-fold higher photoacoustic signals. Micelle preparation was reproducible/scalable, and the CuNC(Octa)-loaded micelles are highly stable under physiological conditions. The CuNC(Octa)-loaded micelles localize within tumors via enhanced permeability and retention and are readily detectable by PA imaging. In a syngeneic murine tumor model of triple-negative breast cancer, CuNC(Octa)-loaded micelles demonstrate efficient heat generation with PTT, leading to the complete eradication of tumors. Conclusions: CuNC(Octa)-loaded micelles represent a promising theranostic agent for PA imaging and PTT. The ability to utilize conventional ultrasound in combination with PA imaging enables the simultaneous acquisition of information about tumor morphology and micelle accumulation. PTT with CuNC(Octa)-loaded micelles can lead to the complete eradication of highly invasive tumors.

Apigenin: a natural molecule at the intersection of sleep and aging.

NAD+, a pivotal coenzyme central to metabolism, exhibits a characteristic decline with age. In mice, NAD+ levels can be elevated via treatment with apigenin, a natural flavonoid that inhibits the NAD+-consuming glycoprotein CD38. In animal models, apigenin positively impacts both sleep and longevity. For example, apigenin improves learning and memory in older mice, reduces tumor proliferation in a mouse xenograft model of triple-negative breast cancer, and induces sedative effects in mice and rats. Moreover, apigenin elongates survival in fly models of neurodegenerative disease and apigenin glycosides increase lifespan in worms. Apigenin's therapeutic potential is underscored by human clinical studies using chamomile extract, which contains apigenin as an active ingredient. Collectively, chamomile extract has been reported to alleviate anxiety, improve mood, and relieve pain. Furthermore, dietary apigenin intake positively correlates with sleep quality in a large cohort of adults. Apigenin's electron-rich flavonoid structure gives it strong bonding capacity to diverse molecular structures across receptors and enzymes. The effects of apigenin extend beyond CD38 inhibition, encompassing agonistic and antagonistic modulation of various targets, including GABA and inflammatory pathways. Cumulatively, a large body of evidence positions apigenin as a unique molecule capable of influencing both aging and sleep. Further studies are warranted to better understand apigenin's nuanced mechanisms and clinical potential.

Improved Antitumor Efficacy of a Dextran-based Docetaxel-coupled Conjugate against Triple-Negative Breast Cancer.

Most chemotherapeutic agents are characterized by poor water solubility and non-specific distribution. Polymer-based conjugates are promising strategies for overcoming these limitations. This study aims to fabricate a polysaccharide, dextran-based, dual-drug conjugate by covalently grafting docetaxel (DTX) and docosahexaenoic acid (DHA) onto the bifunctionalized dextran through a long linker, and to investigate the antitumor efficacy of this conjugate against breast cancer. DTX was firstly coupled with DHA and covalently bounded with the bifunctionalized dextran (100 kDa) through a long linker to produce a conjugate dextran-DHA-DTX (termed C-DDD). Cytotoxicity and cellular uptake of this conjugate were measured in vitro. Drug biodistribution and pharmacokinetics were investigated through liquid chromatography/mass spectrometry analysis. The inhibitory effects on tumor growth were evaluated in MCF-7- and 4T1-tumor-bearing mice. The loading capacity of the C-DDD for DTX was 15.90 (weight/weight). The C-DDD possessed good water solubility and was able to self-assemble into nanoparticles measuring 76.8 ± 5.5 nm. The maximum plasma concentration and area under the curve (0-∞) for the released DTX and total DTX from the C-DDD were significantly enhanced compared with the conventional DTX formulation. The C-DDD selectively accumulated in the tumor, with limited distribution was observed in normal tissues. The C-DDD exhibited greater antitumor activity than the conventional DTX in the triplenegative breast cancer model. Furthermore, the C-DDD nearly eliminated all MCF-7 tumors in nude mice without leading to systemic adverse effects. This dual-drug C-DDD has the potential to become a candidate for clinical application through the optimization of the linker.

Altered gut microbiome drives heightened pain sensitivity in a murine model of metastatic triple-negative breast cancer

Altered gut microbiome drives heightened pain sensitivity in a murine model of metastatic triple-negative breast cancer

Targeting Receptor Tyrosine Kinases as a Novel Strategy for the Treatment of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) comprises a group of aggressive and heterogeneous breast carcinoma. Chemotherapy is the mainstay for the treatment of triple-negative tumors. Nevertheless, the success of chemotherapeutic treatments is limited by their toxicity and development of acquired resistance leading to therapeutic failure and tumor relapse. Hence, there is an urgent need to explore novel targeted therapies for TNBC. Receptor tyrosine kinases (RTKs) are a family of transmembrane receptors that are key regulators of intracellular signaling pathways controlling cell proliferation, differentiation, survival, and motility. Aberrant activity and/or expression of several types of RTKs have been strongly connected to tumorigenesis. RTKs are frequently overexpressed and/or deregulated in triple-negative breast tumors and are further associated with tumor progression and reduced survival in patients. Therefore, targeting RTKs could be an appealing therapeutic strategy for the treatment of TNBC. This review summarizes the current evidence regarding the antitumor activity of RTK inhibitors in preclinical models of TNBC. The review also provides insights into the clinical trials evaluating the use of RTK inhibitors for the treatment of patients with TNBC.

Decanoylcarnitine Inhibits Triple-Negative Breast Cancer Progression via Mmp9 in an Intermittent Fasting Obesity Mouse.

Purpose: Treatment of triple-negative breast cancer (TNBC) remains challenging. Intermittent fasting (IF) has emerged as a promising approach to improve metabolic health of various metabolic disorders. Clinical studies indicate IF is essential for TNBC progression. However, the molecular mechanisms underlying metabolic remodeling in regulating IF and TNBC progression are still unclear. Methods: In this study, we utilized a robust mouse model of TNBC and exposed subjects to a high-fat diet (HFD) with IF to explore its impact on the metabolic reprogramming linked to cancer progression. To identify crucial serum metabolites and signaling events, we utilized targeted metabolomics and RNA sequencing (RNA-seq). Furthermore, we conducted immunoblotting, real-time quantitative polymerase chain reaction (RT-qPCR), cell migration assays, lentivirus-mediated Mmp9 overexpression, and Mmp9 inhibitor experiments to elucidate the role of decanoylcarnitine/Mmp9 in TNBC cell migration. Results: Our observations indicate that IF exerts notable inhibitory effects on both the proliferation and cancer metastasis. Utilizing targeted metabolomics and RNA-seq, we initially identified pivotal serum metabolites and signaling events in the progression of TNBC. Among the 349 serum metabolites identified, decanoylcarnitine was picked out to inhibit TNBC cell proliferation and migration. RNA-seq analysis of TNBC cells treated with decanoylcarnitine revealed its suppressive effects on extracellular matrix-related protein components, with a notable reduction observed in Mmp9. Further investigations confirmed that decanoylcarnitine could inhibit Mmp9 expression in TNBC cells, primary tumors, lung, and liver metastasis tissues. Mmp9 overexpression abolished the inhibitory effect of decanoylcarnitine on cell migration. Conclusion: This study pioneers the exploration of IF intervention and the role of decanoylcarnitine/Mmp9 in the progression of TNBC in obese mice, enhancing our comprehension of the potential roles of various dietary patterns in the process of cancer treatment.

Small-molecule inhibition of kinesin KIF18A reveals a mitotic vulnerability enriched in chromosomally unstable cancers

Chromosomal instability (CIN) is a hallmark of cancer, caused by persistent errors in chromosome segregation during mitosis. Aggressive cancers like high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) have a high frequency of CIN and TP53 mutations. Here, we show that inhibitors of the KIF18A motor protein activate the mitotic checkpoint and selectively kill chromosomally unstable cancer cells. Sensitivity to KIF18A inhibition is enriched in TP53-mutant HGSOC and TNBC cell lines with CIN features, including in a subset of CCNE1-amplified, CDK4–CDK6-inhibitor-resistant and BRCA1-altered cell line models. Our KIF18A inhibitors have minimal detrimental effects on human bone marrow cells in culture, distinct from other anti-mitotic agents. In mice, inhibition of KIF18A leads to robust anti-cancer effects with tumor regression observed in human HGSOC and TNBC models at well-tolerated doses. Collectively, our results provide a rational therapeutic strategy for selective targeting of CIN cancers via KIF18A inhibition.

Targeting Lysosome for Enhanced Cancer Photodynamic/Photothermal Therapy in a "One Stone Two Birds" Pattern.

Highly immunogenic programmed death of tumor cells, such as immunogenic cell death (ICD) and pyroptosis, strengthens antitumor responses and thus represents a promising target for cancer immunotherapy. However, the development of ICD and pyroptosis inducers remains challenging, and their efficiency is typically compromised by self-protective autophagy. Here, we report a potent ICD and pyroptosis-inducing strategy by coupling combined photodynamic/photothermal therapy (PTT/PDT) to biological processes in cancer cells. For this purpose, we rationally synthesize a lysosomal-targeting boron-dipyrromethene dimer (BDPd) with intense NIR absorption/emission, high reactive oxygen species (ROS) yield, and photothermal abilities, which can be self-assembled with Pluronic F127, producing lysosomal-acting nanomicelles (BDPd NPs) to facilitate cancer cell internalization of BDPd and generation of intracellular ROS. Owing to the favorable lysosomal-targeting ability of the morpholine group on BDPd, the intracellular BDPd NPs can accumulate in the lysosome and induce robust lysosomal damage in cancer cells upon 660 nm laser irradiation, which results in the synergetic induction of pyroptosis and ICD via activating NLRP3/GSDMD and caspase-3/GSDME pathways simultaneously. More importantly, PTT/PDT-induced self-protective autophagic degradation was blocked due to the dysfunction of lysosomes. Either intratumorally or intravenously, the injected BDPd NPs could markedly inhibit the growth of established tumor tissues upon laser activation, provoke local and systemic antitumor immune responses, and prolong the survival time in the mouse triple-negative breast cancer model. Collectively, this work represents a promising strategy to boost the therapeutic potential of PTT/PDT by coupling phototherapeutic reagents with the subcellular organelles, creating a "one stone two birds" pattern.

Co-assembly of polymeric conjugates sensitizes neoadjuvant chemotherapy of triple-negative breast cancer with reduced systemic toxicity

Rational design of polymeric conjugates could greatly potentiate the combination therapy of solid tumors. In this study, we designed and prepared two polymeric conjugates (HT-DTX and PEG-YC-1), whereas the drugs were attached to the PEG via a linker sensitive to cathepsin B, over-expressed in TNBC. Stable nanostructures were formed by these two polymer prodrug conjugates co-assembly (PPCC). The stimuli-responsiveness of PPCC was confirmed, and the size shrinkage under tumor microenvironment would facilitate the penetration of PPCC into tumor tissue. In vitro experiments revealed the molecular mechanism for the synergistic effect of the combination of DTX and YC-1. Moreover, the systemic side effects were significantly diminished since the biodistribution of PPCC was improved after i.v. administration in vivo. In this context, the co-assembled nano-structural approach could be employed for delivering therapeutic drugs with different mechanisms of action to exert a synergistic anti-tumor effect against solid tumors, including triple-negative breast cancer. Statement of significance•Co-assembly of polymeric prodrug conjugates could form stable and homogeneous nanostructures, improving cellular uptake by cancer cells and in vivo tumor penetration and retention.•Co-delivery of neoadjuvant therapeutic drug (DTX) and HIF-1α inhibitor (YC-1) synergizes the anti-tumor effect by affecting HIF-1α expression and G2/M phase arrest.•Polymer prodrug conjugates co-assembly (PPCC) could reverse tumor resistance to neoadjuvant therapy, retard tumor progression, and prevent lung metastasis with reduced systemic toxicity in a murine triple-negative breast cancer model.

Apoptosis-driven synergistic anti-cancer efficacy of ethyl acetate extract of Memecylon sisparense Gamble leaves and doxorubicin in in-vitro and in-vivo models of triple-negative breast cancer

In the spectrum of breast neoplasms, approximately 15 to 20% of all diagnosed cases are triple-negative breast carcinoma. TNBC grows and spreads faster than other invasive breast cancers and has a worse prognosis. The existing therapies and chemotherapeutic drugs have several limitations, so the development of safe and affordable treatment options is currently in demand. Hence, this research focuses on scientifically evaluating the therapeutic anticancer effect of ethyl acetate extract of MSG and its combined efficacy with doxorubicin against TNBC. MSG has shown an IC50 value of 48.40±1.68µg/ml on the MDA-MB-231 cell line, and the combination of MSG with Dox demonstrated the synergistic effect. Apoptotic changes such as membrane blebbing chromatin condensation were observed in MSG alone and in combination with doxorubicin treatments. Apoptosis was confirmed with Annexin V-FITC/PI staining and increased apoptotic markers such as Cleaved caspase-3 Bax and decreased anti-apoptotic markers Bcl-2 by western blotting. The tumor burden significantly decreased in MSG and combination treatment groups while restoring their body weights. Meanwhile, the Dox-treated group indicated a decreased tumor burden combined with weight loss. The present investigation revealed that MSG and doxorubicin have a synergistic anticancer effect in TNBC.

Development of a HPLC-MS/MS Method to Assess the Pharmacokinetics and Tumour Distribution of the Dimethylarginine Dimethylaminohydrolase 1 Inhibitors ZST316 and L-257 in a Xenograft Model of Triple-Negative Breast Cancer in Mice.

We describe the development and validation of an HPLC-MS/MS method to assess the pharmacokinetics and tumour distribution of ZST316, an arginine analogue with inhibitory activity towards dimethylarginine dimethylaminohydrolase 1 (DDAH1) and vasculogenic mimicry, and its active metabolite L-257 in a xenograft model of triple-negative breast cancer (TNBC). The method proved to be reproducible, precise, and highly accurate for the measurement of both compounds in plasma and tumour tissue following acute and chronic (five days) intraperitoneal administration of ZST316 (30 mg/Kg daily) in six-week-old severe combined immunodeficiency disease (SCID) mice inoculated with MDA-MB-231 TNBC cells. ZST316 was detected in tumour tissue and plasma after 1 h (6.47 and 9.01 μM, respectively) and 24 h (0.13 and 0.16 μM, respectively) following acute administration, without accumulation during chronic treatment. Similarly, the metabolite L-257 was found in tumour tissue and plasma after 1 h (15.06 and 8.72 μM, respectively) and 24 h (0.17 and 0.17 μM, respectively) following acute administration of ZST316, without accumulation during chronic treatment. The half-life after acute and chronic treatment ranged between 4.4-7.1 h (plasma) and 4.5-5.0 h (tumour) for ZST316, and 4.2-5.3 h (plasma) and 3.6-4.9 h (tumour) for L-257. The results of our study demonstrate the (a) capacity to accurately measure ZST316 and L-257 concentrations in plasma and tumour tissue in mice using the newly developed HPLC-MS/MS method, (b) rapid conversion of ZST316 into L-257, (c) good intra-tumour penetration of both compounds, and (d) lack of accumulation of both ZST316 and L-257 in plasma and tumour tissue during chronic administration. Compared to a previous method developed by our group to investigate ZST316 in plasma, the main advantages of the new method include a wider range of linearity which reduces the need for dilutions and the combined assessment of ZST316 and L-257 in plasma and tumour tissue which limits the required amount of matrix. The new HPLC-MS/MS method is useful to investigate the in vivo effects of ZST316 and L-257 on vasculogenic mimicry, tumour mass, and metastatic burden in xenograft models of TNBC.

PD-L1 blockade TAM-dependently potentiates mild photothermal therapy against triple-negative breast cancer

The present work was an endeavor to shed light on how mild photothermia possibly synergizes with immune checkpoint inhibition for tumor therapy. We established mild photothermal heating protocols to generate temperatures of 43 °C and 45 °C in both in vitro and in vivo mouse 4T1 triple-negative breast cancer (TNBC) models using polyglycerol-coated carbon nanohorns (CNH-PG) and 808 nm laser irradiation. Next, we found that 1) CNH-PG-mediated mild photothermia (CNH-PG-mPT) significantly increased expression of the immune checkpoint PD-L1 and type-1 macrophage (M1) markers in the TNBC tumors; 2) CNH-PG-mPT had a lower level of anti-tumor efficacy which was markedly potentiated by BMS-1, a PD-L1 blocker. These observations prompted us to explore the synergetic mechanisms of CNH-PG-mPT and BMS-1 in the context of tumor cell-macrophage interactions mediated by PD-L1 since tumor-associated macrophages (TAMs) are a major source of PD-L1 expression in tumors. In vitro, the study then identified two dimensions where BMS-1 potentiated CNH-PG-mPT. First, CNH-PG-mPT induced PD-L1 upregulation in the tumor cells and showed a low level of cytotoxicity which was potentiated by BMS-1. Second, CNH-PG-mPT skewed TAMs towards an M1-like anti-tumor phenotype with upregulated PD-L1, and BMS-1 bolstered the M1-like phenotype. The synergistic effects of BMS-1 and CNH-PG-mPT both on the tumor cells and TAMs were more pronounced when the two cell populations were in co-culture. Further in vivo study confirmed PD-L1 upregulation both in tumor cells and TAMs in the TNBC tumors following treatment of CNH-PG-mPT. Significantly, TAMs depletion largely abolished the anti-TNBC efficacy of CNH-PG-mPT alone and in synergy with BMS-1. Collectively, our findings reveal PD-L1 upregulation to be a key response of TNBC to mild photothermal stress, which plays a pro-survival role in the tumor cells while also acting as a brake on the M1-like activation of the TAMs. Blockade of mPT‑induced PD‑L1 achieves synergistic anti-TNBC efficacy by taking the intrinsic survival edge off the tumor cells on one hand and taking the brakes off the M1-like TAMs on the other. Our findings reveal a novel way (i.e. mild thermia plus PD-L1 blockade) to modulate the TAMs-tumor cell interaction to instigate a mutiny of the TAMs against their host tumor cells.Graphical

Type-I protein arginine methyltransferase inhibition primes anti-programmed cell death protein 1 immunotherapy in triple-negative breast cancer.

Immune-checkpoint blockade (ICB) therapy shows promise for treating aggressive triple-negative breast cancer (TNBC). However, only some patients benefit from ICB, revealing an urgent need for identifying novel strategies for sensitizing patients to ICB. Previously, the authors demonstrated that type-I protein arginine methyltransferases (PRMTs) regulated antiviral innate-immune responses in TNBC by altering RNA splicing. This study aimed to explore the effects of targeting type-I PRMTs on the tumor microenvironment (TME) and the efficacy of ICB therapy against TNBC. Single-cell transcriptomic analysis was performed to investigate the effects of type-I PRMT inhibition on the TME, especially T-cell subsets. Single-cell T-cell receptor sequencing was performed to analyze the diversity and dynamics of the T-cell repertoire. A syngeneic murine model of TNBC was used to evaluate the therapeutic efficacy and immune memory effect of combining a type-I PRMT inhibitor (MS023) with an anti-programmed cell death protein 1 (PD-1) antibody. Type-I PRMT inhibition combined with anti-PD-1 therapy reduced tumor growth. Mechanistically, type-I PRMT inhibition reshaped the TME. Increased CD8 T-cell infiltration was verified using flow cytometry. Increased clonotypes and clonal diversity were also observed after MS023 treatment, which contributed to immune memory following combination treatment. Targeting type-I PRMT can potentially improve immunotherapeutic efficacies in patients with TNBC. By enhancing the tumor immunogenicity and promoting a more favorable immune microenvironment, this combined approach may enable more patients with TNBC to benefit from immunotherapies.

Amplifying cancer chemoimmunotherapy through F4/80+CD11c+ DC-like macrophages induced by micellar docetaxel together with a TLR7/8 nanoagonist

Chemotherapy-based combinational immunomodulation has been developed to inflame the “cold” tumor immune microenvironment (TiME) for enhanced triple-negative breast cancer (TNBC) chemoimmunotherapy. However, the potential of docetaxel-based chemotherapy combined with immunomodulation by TLR7/8 agonists remains undiscovered. Herein, we first constructed micellar docetaxel (DTX-m) and a TLR7/8 nanoagonist suitable for systematic administration, R848 nanoparticles (R848 NPs). By adjusting the administration sequence, the combination of DTX-m and R848 NPs can efficiently inhibit tumor growth in vivo in a rodent triple-negative breast cancer model. The results of a mechanistic study reveal that alternate sequential administration promotes the polarization of macrophages to F4/80+CD11c+ DC-like macrophages and cumulatively promotes the secretion of CXCL10 and proinflammatory cytokines, which boost the recruitment of NK cells and APCs to reinflame the tumor immune microenvironment, further facilitating the recruitment and proliferation of effector T cells and amplifying tumor chemoimmunotherapy. Administration sequence regulates the therapeutic outcome by steering the polarization of macrophage phenotypes.

Abstract LB_C06: Utilizing a novel HDAC inhibitor bocodepsin (OKI-179) to overcome doxorubicin resistance in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic treatment options and a high rate of metastatic recurrence. TNBC is defined by lack of estrogen and progesterone receptor expression with non-amplified HER2, and accounts for 10-15% of all breast cancer cases. While pembrolizumab, sacituzumab govitecan and PARP inhibitors are beneficial for a subset of TNBC patients, chemotherapy including doxorubicin continues to be the primary treatment. Bocodepsin is a novel, orally bioavailable clinical stage Class 1-targeting HDAC inhibitor with activity in preclinical models of TNBC. The purpose of this study was to investigate OKI-005 (in vitro) and bocodepsin (in vivo) in combination with doxorubicin (DOX) to overcome resistance. Procedures: TNBC cell lines were exposed to a no drug, DOX, OKI-005 or combination for 72 hours and cell viability was assessed via CellTiter-Glo. Synergy scores were calculated from three independent replicates using Synergy Finder+. Apoptosis was assessed following 48 hours of drug exposure by flow cytometry. Immunoblots were performed following 24 hours of drug treatment to evaluate mechanism of action. In vitro senescence-associated beta-galactosidase was performed after 6 days drug treatment. For in vivo studies, 5 million MDA-MB-231 cells were injected subcutaneously in athymic nude mice. Mice were treated with either no drug, 1.5 mg/kg DOX IP QW, 80 mg/kg bocodepsin PO QD, or the combination. Results: Bliss scores calculated from Synergy Finder+ using the CellTiter-Glo data showed synergy for the combination of OKI-005 and DOX in all TNBC cell lines. The combination resulted in statistically significant increased apoptosis measured by Annexin V compared to single agent drug treatment in CAL-51, MDA-MB-231 and CAL-120 cell lines. Immunoblot analysis demonstrated an increase in the pro-apoptotic protein BIM and decrease in anti-apoptotic protein BCL-XL with the combination. The cyclin dependent kinase inhibitor p21 was increased by OKI-005 independently of p53 and doxorubicin for the cell lines MDA-MB-231, Hs 578T, and CAL-120. The combination resulted in increased cleaved caspase-3 for all cell lines evaluated. Senescence-associated beta-galactosidase showed an increase in senescent cells for both DOX and OKI-005, which was eradicated in combination. DOX and bocodepsin were further tested in vivo, where significant tumor growth inhibition was observed in combination compared to DOX (p=0.0016) and bocodepsin (p=0.0068). Conclusion: The addition of OKI-005 or bocodepsin to doxorubicin resulted in improved anti-proliferative and anti-cancer activity with evidence of increased apoptosis and decreased senescence. As a result, bocodepsin is a promising combination partner for doxorubicin to overcome doxorubicin resistance and promote apoptosis in TNBC. Bocodepsin has a favorable toxicity profile compared to other HDAC inhibitors and this work supports the potential clinical investigation of doxorubicin and bocodepsin in patients with metastatic TNBC. Citation Format: Stephen G Smoots, Anna R Schreiber, Marilyn M Jackson, Evan D Dus, Stacey M Bagby, Adrian T A. Dominguez, Cameron A Binns, Jennifer R Diamond, Todd M Pitts. Utilizing a novel HDAC inhibitor bocodepsin (OKI-179) to overcome doxorubicin resistance in triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C06.