Metastatic Breast Cancer Model Research Articles

Resistance to mesenchymal reprogramming sustains clonal propagation in metastatic breast cancer

The acquisition of mesenchymal traits is considered a hallmark of breast cancer progression. However, the functional relevance of epithelial-to-mesenchymal transition (EMT) remains controversial and context dependent. Here, we isolate epithelial and mesenchymal populations from human breast cancer metastatic biopsies and assess their functional potential invivo. Strikingly, progressively decreasing epithelial cell adhesion molecule (EPCAM) levels correlate with declining disease propagation. Mechanistically, we find that persistent EPCAM expression marks epithelial clones that resist EMT induction and propagate competitively. In contrast, loss of EPCAM defines clones arrested in a mesenchymal state, with concomitant suppression of tumorigenicity and metastatic potential. This dichotomy results from distinct clonal trajectories impacting global epigenetic programs that are determined by the interplay between human ZEB1 and its target GRHL2. Collectively, our results indicate that susceptibility to irreversible EMT restrains clonal propagation, whereas resistance to mesenchymal reprogramming sustains disease spread in multiple models of human metastatic breast cancer, including patient-derived cells invivo.

Abstract LB347: A lung-mimicking synthetic metastatic niche reveals N1 neutrophils drive breast cancer metastatic dormancy in the lungs

Abstract 3D scaffolds mimicking the environment in the primary tumor or metastatic organs can deconstruct complex niche signals and facilitate the study of cancer progression and metastasis. Here, we reported that a subcutaneous 3D scaffold implant acted as a lung-mimicking dormant metastatic niche in mouse models of metastatic breast cancer, recruiting lung-tropic circulating tumor cells yet suppressing their growth through potent in situ antitumor immunity. We compared it with the immunosuppressive lungs developing lethal metastases and the dormant lungs suppressing tumor growth derived from breast cancer models with varying tumor aggressiveness and host immunity. Our data suggested that breast cancer-induced Gr1+CD11b+Ly6G+ granulocytic myeloid cells (neutrophils) infiltrated the scaffold implants and lungs, secreting the same signal to facilitate the metastatic seeding of lung-tropic cancer cells in these two types of niches. However, circulating neutrophils with opposing phenotypes and functions (N1 and N2) were selectively recruited and enriched in the dormant scaffolds/lungs and immunosuppressive lungs, respectively, responding to two distinct groups of chemoattractants. N1 or N2 neutrophils established activated or suppressive immune environments in the metastatic niches, directing different fates of cancer cells. The clinical relevance of these scientific findings was validated by the strong positive correlation of a high N1-to-N2 neutrophil chemoattractant ratio with a low-grade primary tumor, a low metastases incidence, and a better prognosis in breast cancer patients. Overall, our study revealed the multifaceted roles of neutrophils in regulating lung metastasis and underscored the importance of N1 neutrophils in driving breast cancer metastatic dormancy in the lungs, inspiring next-generation immunotherapy. Citation Format: Jing Wang, Ramon Ocadiz-Ruiz, Matthew Hall, Grace Bushnell, Sophia Orbach, Joseph Decker, Ravi Raghani, Yining Zhang, Aaron Morris, Jacqueline Jeruss, Lonnie Shea. A lung-mimicking synthetic metastatic niche reveals N1 neutrophils drive breast cancer metastatic dormancy in the lungs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB347.

Neoadjuvant immune checkpoint blockade triggers persistent and systemic Treg activation which blunts therapeutic efficacy against metastatic spread of breast tumors

ABSTRACT The clinical successes of immune checkpoint blockade (ICB) in advanced cancer patients have recently spurred the clinical implementation of ICB in the neoadjuvant and perioperative setting. However, how neoadjuvant ICB therapy affects the systemic immune landscape and metastatic spread remains to be established. Tumors promote both local and systemic expansion of regulatory T cells (Tregs), which are key orchestrators of tumor-induced immunosuppression, contributing to immune evasion, tumor progression and metastasis. Tregs express inhibitory immune checkpoint molecules and thus may be unintended targets for ICB therapy counteracting its efficacy. Using ICB-refractory models of spontaneous primary and metastatic breast cancer that recapitulate the poor ICB response of breast cancer patients, we observed that combined anti-PD-1 and anti-CTLA-4 therapy inadvertently promotes proliferation and activation of Tregs in the tumor, tumor-draining lymph node and circulation. Also in breast cancer patients, Treg levels were elevated upon ICB. Depletion of Tregs during neoadjuvant ICB in tumor-bearing mice not only reshaped the intratumoral immune landscape into a state favorable for ICB response but also induced profound and persistent alterations in systemic immunity, characterized by elevated CD8+ T cells and NK cells and durable T cell activation that was maintained after treatment cessation. While depletion of Tregs in combination with neoadjuvant ICB did not inhibit primary tumor growth, it prolonged metastasis-related survival driven predominantly by CD8+ T cells. This study demonstrates that neoadjuvant ICB therapy of breast cancer can be empowered by simultaneous targeting of Tregs, extending metastasis-related survival, independent of a primary tumor response.

Abstract 548: The microRNA-10b targeted therapeutic, TTX-MC138, is effective in preclinical pancreatic adenocarcinoma

Abstract Non-coding RNAs, such as miRNAs, represent promising targets for cancer therapy, given their role as critical players in oncogenic processes, including tumor initiation and progression, metastasis and therapeutic resistance. We and others demonstrated that miRNA-10b is a critical driver of tumor cell viability, especially in the metastatic setting. Based on this discovery, it became feasible to treat established metastatic cancer by inhibiting miR-10b. This was accomplished using a therapeutic, termed TTX-MC138, composed of antagomirs against miR-10b, conjugated to dextran-coated iron oxide nanoparticles. In our previous studies in murine models of metastatic breast cancer, we demonstrated the effectiveness of TTX-MC138 at preventing and eliminating existing metastases. Here, we describe the application of TTX-MC138 for the treatment of pancreatic adenocarcinoma. TTX-MC138 was administered weekly via intravenous injection into mice bearing orthotopic xenografts derived from human pancreatic adenocarcinoma cells. Tumor burden was measured by in vivo bioluminescence imaging. Animal survival, body weight, and tumor and metastatic burden at necropsy were also analyzed. Serum and tumor miR-10b expression, as well as global gene expression using RNA-seq were assessed to provide insight into target engagement and molecular function. Animals treated with vehicle or gemcitabine served as controls. Tumor growth rate was found to be significantly lower in animals treated with TTX-MC138 vs controls (p < 0.0001 for TTX-MC138 vs. PBS and vs. gemcitabine). Importantly, 40% of the animals treated with TTX-MC138 regressed their tumors completely as measured by bioluminescence imaging. Treatment continued for 10 weeks, after which the responding mice were monitored for disease recurrence without treatment for an additional 10 weeks. The animals showed evidence of continued weight gain and durable survival. They were sacrificed 10 weeks after treatment was discontinued, despite no evidence of morbidity, to perform necropsy. No tumors or metastases were found at the time of sacrifice. Gene expression analysis confirmed target engagement and molecular mechanism of action. Against the background of our earlier work, these studies provide further evidence of the potential of TTX-MC138 for cancer therapy and expand its relevance beyond metastatic breast cancer to also include pancreatic adenocarcinoma. Citation Format: Subrata Ghosh, Douglas Lazarus, Neil Robertson, Edward Crosier, Peter Liu, Zdravka Medarova. The microRNA-10b targeted therapeutic, TTX-MC138, is effective in preclinical pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 548.

Abstract 5167: Entinostat's modulation of myeloid derived suppressor cells through the STAT3-NFκB-AP-1 axis decreases suppressive signaling

Abstract Metastatic breast cancer remains one of the leading causes of global cancer incidence in women, despite the benefit of immune checkpoint inhibitors (ICIs) in managing various cancers and improving patient quality of life. Metastatic breast cancer is characterized by extensive infiltration of the tumor microenvironment (TME) with immunosuppressive cells, such as myeloid derived suppressor cells (MDSCs), that inhibit anti-tumoral immune cells and prevent effective activation of the adaptive immune system by ICIs. Previously, our group has shown in the breast TME that epigenetic reprogramming of MDSCs by entinostat, a histone deacetylase inhibitor, decreases MDSC immunosuppressive function and enhances response to ICIs, in part mediated by altered signaling within the Signal transducer and activator of transcription 3 (STAT3) and Nuclear factor kappa B (NFκB) axis. Next, we sought to examine the effects of entinostat on MDSC reprogramming in a distant metastatic TME. Using the syngeneic NT2.5LM NeuN mouse model of metastatic breast cancer, we established spontaneous lung metastases and treated with either vehicle or entinostat (5 mg/kg by oral gavage, 5x/week) for 3 weeks. Single cell RNA sequencing (scRNAseq) of macro-dissected lung metastases revealed a large MDSC population, and unsupervised pathways analysis of the MDSC cluster showed differential regulation of the IL6-JAK-STAT3 and TNFα-signaling-via-NFκB pathways upon entinostat treatment. In line with reports of crosstalk among STAT3, NFκB, and AP-1 pathways regulating inflammation in breast cancer, we also found through scRNAseq differential expression of AP-1 subunits JunB and FOSL1 upon entinostat treatment. At the protein level, western blot analysis of isolated intratumoral MDSCs from lung metastases revealed decreased STAT3 phosphorylation upon entinostat treatment. Using J774M cells, an MDSC-like cell line, we found decreased JunB phosphorylation and decreased FOSL1 protein upon entinostat treatment. Furthermore, preliminary evaluation of imaging mass cytometry (IMC) from tumor biopsies in selected patients with metastatic breast cancer treated with entinostat during the clinical trial NCI-9844 confirmed decreased STAT3 phosphorylation in MDSCs. Taken together, we provide new evidence in the metastatic lung TME that implicates a STAT3-NFκB-AP-1 mediated mechanism leading to decreased MDSC suppression. Evaluating this mechanism in MDSCs taken directly from treated lung metastases represents the most biologically relevant mechanistic study to date, and preliminary translation of findings in patients suggests that planned studies will lead to identification of the mechanism driving response. Citation Format: Aaron G. Baugh, Edgar Gonzalez, Valerie H. Narumi, Sofi Castanon, Jesse Kreger, James Leatherman, Won Jin Ho, Ashley Cimino-Mathews, Vered Stearns, Roisin M. Connolly, Elizabeth M. Jaffee, Adam L. MacLean, Evanthia T. Roussos Torres. Entinostat's modulation of myeloid derived suppressor cells through the STAT3-NFκB-AP-1 axis decreases suppressive signaling. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5167.

New Method for Counting and Picking Out Single Circulating Tumor Cells from Microliter-Volume Samples for Tumor Progression Surveillance and Single-Cell Heterogeneity Analysis

Circulating tumor cells (CTCs) are crucial in tumor progression and metastasis, but the knowledge of their roles grows slowly at single-cell levels. Characterizing the rarity and fragility of CTCs by nature, highly stable and efficient single-CTC sampling methods are still lacking, which impedes the development of single-CTC analysis. Herein, an improved, capillary-based single-cell sampling (SiCS) method, the so-called bubble-glue single-cell sampling (bubble-glue SiCS), is introduced. Benefiting from the characteristic that the cells tend to adhere to air bubbles in the solution, single cells can be sampled with bubbles as low as 20 pL with a self-designed microbubble-volume-controlled system. Benefiting from the excellent maneuverability, single CTCs are sampled directly from 10 μL volume of real blood samples after fluorescent labeling. Meanwhile, over 90% of the CTCs obtained survived and well proliferated after the bubble-glue SiCS process, which showed considerable superiority for downstream single-CTC profiling. Furthermore, a highly metastatic breast cancer model of the 4T1 cell line in vivo was employed for the real blood sample analysis. Increases in CTC numbers were observed during the tumor progression process, and significant heterogeneities among individual CTCs were discovered. In all, we propose a novel avenue for target SiCS and provide an alternative technique route for CTC separation and analysis.

Peptide‐Decorated Artificial Erythrocyte Microvesicles Endowed with Lymph Node Targeting Function for Drug Delivery

AbstractMicrovesicles (MVs) are heterogeneous membrane‐bound sacs formed by direct plasma membrane budding, that have promising potential for targeted drug delivery and therapy. However, to date, they have limited application to lymph node (LN) targeting due to their large size. Herein, the direct size conversion of erythrocyte‐derived MVs (eMVs) via incubation with an ApoA‐I mimetic peptide (R4F) capable of binding phospholipids is performed. The interaction between the R4F peptide and phospholipid bilayers of eMVs limits the maximum possible length of the lipid‐bound conformation and induces the formation of small‐sized eMVs, which further endows them with SR‐B1 receptor and LN targeting functions. In a lymphadenitis model, dexamethasone (Dex)‐loaded eMVs functionalized with R4F peptide can specifically suppress the macrophage inflammatory response through the NF‐κB and p38 MAPK pathways along with reducing inflammatory cell infiltration to improve lymphadenitis. Moreover, celastrol (Cel)‐loaded eMVs functionalized with R4F peptide can significantly inhibit LN metastasis by selectively killing SR‐B1–overexpressing 4T1 tumor cells and inducing T cell‐mediated systemic antitumor immune responses in LN metastatic model of breast cancer. Thus, small R4F peptide‐decorated eMVs with SR‐B1–targeting ability provide an excellent drug delivery system for the targeted treatment of LN‐associated diseases, opening new frontiers for LN‐targeted drug delivery.

Evaluation of Alisertib Alone or Combined With Fulvestrant in Patients With Endocrine-Resistant Advanced Breast Cancer

Aurora A kinase (AURKA) activation, related in part to AURKA amplification and variants, is associated with downregulation of estrogen receptor (ER) α expression, endocrine resistance, and implicated in cyclin-dependent kinase 4/6 inhibitor (CDK 4/6i) resistance. Alisertib, a selective AURKA inhibitor, upregulates ERα and restores endocrine sensitivity in preclinical metastatic breast cancer (MBC) models. The safety and preliminary efficacy of alisertib was demonstrated in early-phase trials; however, its activity in CDK 4/6i-resistant MBC is unknown. To assess the effect of adding fulvestrant to alisertib on objective tumor response rates (ORRs) in endocrine-resistant MBC. This phase 2 randomized clinical trial was conducted through the Translational Breast Cancer Research Consortium, which enrolled participants from July 2017 to November 2019. Postmenopausal women with endocrine-resistant, ERBB2 (formerly HER2)-negative MBC who were previously treated with fulvestrant were eligible. Stratification factors included prior treatment with CDK 4/6i, baseline metastatic tumor ERα level measurement (<10%, ≥10%), and primary or secondary endocrine resistance. Among 114 preregistered patients, 96 (84.2%) registered and 91 (79.8%) were evaluable for the primary end point. Data analysis began after January 10, 2022. Alisertib, 50 mg, oral, daily on days 1 to 3, 8 to 10, and 15 to 17 of a 28-day cycle (arm 1) or alisertib same dose/schedule with standard-dose fulvestrant (arm 2). Improvement in ORR in arm 2 of at least 20% greater than arm 1 when the expected ORR for arm 1 was 20%. All 91 evaluable patients (mean [SD] age, 58.5 [11.3] years; 1 American Indian/Alaskan Native [1.1%], 2 Asian [2.2%], 6 Black/African American [6.6%], 5 Hispanic [5.5%], and 79 [86.8%] White individuals; arm 1, 46 [50.5%]; arm 2, 45 [49.5%]) had received prior treatment with CDK 4/6i. The ORR was 19.6%; (90% CI, 10.6%-31.7%) for arm 1 and 20.0% (90% CI, 10.9%-32.3%) for arm 2. In arm 1, the 24-week clinical benefit rate and median progression-free survival time were 41.3% (90% CI, 29.0%-54.5%) and 5.6 months (95% CI, 3.9-10.0), respectively, and in arm 2 they were 28.9% (90% CI, 18.0%-42.0%) and 5.4 months (95% CI, 3.9-7.8), respectively. The most common grade 3 or higher adverse events attributed to alisertib were neutropenia (41.8%) and anemia (13.2%). Reasons for discontinuing treatment were disease progression (arm 1, 38 [82.6%]; arm 2, 31 [68.9%]) and toxic effects or refusal (arm 1, 5 [10.9%]; arm 2, 12 [26.7%]). This randomized clinical trial found that adding fulvestrant to treatment with alisertib did not increase ORR or PFS; however, promising clinical activity was observed with alisertib monotherapy among patients with endocrine-resistant and CDK 4/6i-resistant MBC. The overall safety profile was tolerable. ClinicalTrials.gov Identifier: NCT02860000.

Abstract P3-08-02: Prospective Use of Feline Mammary Carcinomas to Study MiR-10b and Metastasis

Abstract Despite many advances in cancer treatment, metastatic disease is estimated to be responsible for 90% of all cancer-related deaths. Current treatments for metastatic disease target various aspects of carcinogenesis but not specifically the metastatic process, representing a major unmet clinical need. MicroRNA-10b, a small non-coding RNA, offers tremendous potential as a treatment target for metastatic disease. It is implicated in invasion, migration, and viability of metastatic cells across a variety of cancer types, and it is upregulated in metastases compared to their matched primary tumor, establishing miR-10b as a potential treatment target unique to metastatic niche. We have previously developed a therapeutic targeting miR-10b in metastases and tested it in murine models of metastatic breast cancer. This therapeutic, consisting of an anti-miR-10b antisense oligonucleotide conjugated to iron oxide-based nanoparticles, prevents metastasis and eradicates pre-existing metastases in murine breast cancer models. With an outlook to clinical translation of our approach, we seek to test our therapeutic strategy in larger animal models. Feline mammary carcinomas are considered by most to be the best large animal model for human breast cancer due to similarities such as relative age of onset, histopathology, metastatic patterns, and treatment response. To support the use of this model with our therapeutic, we investigated the characteristics of miR-10b expression in spontaneous metastatic breast cancer in companion cats. Archival blocks of matched primary tumors and metastatic lymph nodes from companion cats diagnosed with mammary carcinoma (n=9, 44%TNBC, 56%HER2+) were obtained from the tissue bank of the Michigan State University (MSU) Veterinary Diagnostic Laboratory (VDL). Tissues were analyzed for miR-10b and its target HOXD10 expression using qRT-PCR and in situ hybridization. qRT-PCR revealed that miR-10b expression was significantly upregulated in 55.5% of lymph node metastases compared to their matched primary tumor, mirroring findings in human metastatic cancer. This was validated by qRT-PCR for HOXD10 gene expression (a direct target of miR-10b), which was significantly downregulated in these metastases compared to their matched primary tumor. In situ hybridization demonstrated that miR-10b expression was increased at the invasive edge of tumors and in actively invading cells, suggesting miR-10b plays a similar role in invasion in feline breast cancer as it does in human breast cancer. Altogether, these findings support the use of feline mammary carcinomas as a model of human breast cancer and as an excellent candidate for treatment with our therapeutic. Citation Format: Alan Halim, N. Anna Savan, Paulo Vilar Saavedra, Vilma Yuzbasiyan-Gurkan, Matti Kiupel, Lorenzo Sempere, Anna Moore. Prospective Use of Feline Mammary Carcinomas to Study MiR-10b and Metastasis [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 P3-08-02.

Immunosuppressive reprogramming of neutrophils by lung mesenchymal cells promotes breast cancer metastasis.

Neutrophils, the most abundant innate immune cells, function as crucial regulators of the adaptive immune system in diverse pathological conditions, including metastatic cancer. However, it remains largely unknown whether their immunomodulatory functions are intrinsic or acquired within the pathological tissue environment. Here, using mouse models of metastatic breast cancer in the lungs, we show that, although neutrophils isolated from bone marrow (BM) or blood are minimally immunosuppressive, lung-infiltrating neutrophils are robustly suppressive of both T cells and natural killer (NK) cells. We found that this tissue-specific immunosuppressive capacity of neutrophils exists in the steady state and is reinforced by tumor-associated inflammation. Acquisition of potent immunosuppression activity by lung-infiltrating neutrophils was endowed by the lung-resident stroma, specifically CD140a+ mesenchymal cells (MCs) and largely via prostaglandin-endoperoxide synthase 2 (PTGS2), the rate-limiting enzyme for prostaglandin E2 (PGE2) biosynthesis. MC-specific deletion of Ptgs2 or pharmacological inhibition of PGE2 receptors reversed lung neutrophil-mediated immunosuppression and mitigated lung metastasis of breast cancer in vivo. These lung stroma-targeting strategies substantially improved the therapeutic efficacy of adoptive T cell-based immunotherapy in treating metastatic disease in mice. Collectively, our results reveal that the immunoregulatory effects of neutrophils are induced by tissue-resident stroma and that targeting tissue-specific stromal factors represents an effective approach to boost tissue-resident immunity against metastatic disease.

Heterodimeric IL-15 (hetIL-15) reduces circulating tumor cells and metastasis formation improving chemotherapy and surgery in 4T1 mouse model of TNBC.

Immunotherapy has emerged as a viable approach in cancer therapy, with cytokines being of great interest. Interleukin IL-15 (IL-15), a cytokine that supports cytotoxic immune cells, has been successfully tested as an anti-cancer and anti-metastatic agent, but combinations with conventional chemotherapy and surgery protocols have not been extensively studied. We have produced heterodimeric IL-15 (hetIL-15), which has shown anti-tumor efficacy in several murine cancer models and is being evaluated in clinical trials for metastatic cancers. In this study, we examined the therapeutic effects of hetIL-15 in combination with chemotherapy and surgery in the 4T1 mouse model of metastatic triple negative breast cancer (TNBC). hetIL-15 monotherapy exhibited potent anti-metastatic effects by diminishing the number of circulating tumor cells (CTCs) and by controlling tumor cells colonization of the lungs. hetIL-15 treatment in combination with doxorubicin resulted in enhanced anti-metastatic activity and extended animal survival. Systemic immune phenotype analysis showed that the chemoimmunotherapeutic regimen shifted the tumor-induced imbalance of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in favor of cytotoxic effector cells, by simultaneously decreasing PMN-MDSCs and increasing the frequency and activation of effector (CD8+T and NK) cells. Tumor resection supported by neoadjuvant and adjuvant administration of hetIL-15, either alone or in combination with doxorubicin, resulted in the cure of approximately half of the treated animals and the development of anti-4T1 tumor immunity. Our findings demonstrate a significant anti-metastatic potential of hetIL-15 in combination with chemotherapy and surgery and suggest exploring the use of this regimen for the treatment of TNBC.

Misaligned Chromosomes are a Major Source of Chromosomal Instability in Breast Cancer.

We surveyed the single-cell landscape of mitotic defects that generate CIN in primary and metastatic breast cancer and relevant models. Misaligned chromosomes predominate, and are less immunostimulatory than other chromosome segregation errors.

Pipeline to identify neoantigens exposed by radiation.

Mutation-associated neoantigens are key targets of tumor-specific T cells and thus play a major role in driving responses to immune checkpoint blockade (ICB) therapy in tumors with high mutational burden. However, only a small number of mutated peptides are actually presented by MHC molecules and only a minority can induce T cell responses. In addition, the recognition of these neoantigens by T cells is limited by the level of expression of the mutated gene product in the tumor cells. Preclinical studies have shown that radiation can convert the irradiated tumor into an in situ vaccine, leading to the priming of tumor-specific T cells and to the rejection of otherwise ICB-resistant tumors. There is now preclinical and clinical evidence that radiation can upregulate the expression of genes containing immunogenic mutations and expose them to the immune system. Therefore, the identification of neoantigens upregulated by radiation could help to predict which patients might benefit from treatment with combinations of radiotherapy and ICB and could also be incorporated into personalized neoantigen vaccination strategies. In this chapter, we present the pipeline that we used to identify relevant radiation-upregulated neoantigens in a poorly immunogenic mouse model of metastatic breast cancer.

The anti-breast cancer stem cell properties of gold(i)-non-steroidal anti-inflammatory drug complexes.

The anti-breast cancer stem cell (CSC) properties of a series of gold(i) complexes comprising various non-steroidal anti-inflammatory drugs (NSAIDs) and triphenylphosphine 1-8 are reported. The most effective gold(i)-NSAID complex 1, containing indomethacin, exhibits greater potency for breast CSCs than bulk breast cancer cells (up to 80-fold). Furthermore, 1 reduces mammosphere viability to a better extent than a panel of clinically used breast cancer drugs and salinomycin, an established anti-breast CSC agent. Mechanistic studies suggest 1-induced breast CSC death results from breast CSC entry, cytoplasm localisation, an increase in intracellular reactive oxygen species levels, cyclooxygenase-2 downregulation and inhibition, and apoptosis. Remarkably, 1 also significantly inhibits tumour growth in a murine metastatic triple-negative breast cancer model. To the best of our knowledge, 1 is the first gold complex of any geometry or oxidation state to demonstrate anti-breast CSC properties.

Detection of circulating tumor cells using antibody-functionalized microchips to monitor tumorigenesis in a mouse model of metastatic breast cancer

Circulating tumor cell (CTC)-based liquid biopsy has been a promising and potentially informative diagnostic approach. However, its clinical application has remained limited until now, in part due to the lack of systematic research to validate the new technologies for CTC enrichment. In this work, we established a 4T1 mouse model of breast cancer recurrence after surgery to evaluate microfluidic-based CTC detection and monitor tumor recurrence. The time point detected by CTC analysis using EpCAM antibody-functionalized chip was first compared with in vivo bioimaging and pathological examination for monitoring metastasis. The CTCs were monitored after tumor resection to evaluate CTC detection for postoperative monitoring of tumor recurrence. Using an antibody-functionalized chip, CTCs can be detected 10-days earlier than being detected with other imaging technologies. The CTC detection assay also showed that EpCAM+ CTC populations increased with tumor growth, whereas EpCAM+ CSV+ CTCs increased first and then decreased, possibly indicating multistep tumorigenesis in this model. Monitoring of CTCs after tumor resection indicated that CTCs were gradually cleared from the blood during mice recovery. These results provide a basis for CTC detection to monitor tumorigenesis and metastasis, as well as a model for validating other liquid biopsy methods.

Endothelial-mesenchymal transition induced by metastatic 4T1 breast cancer cells in pulmonary endothelium in aged mice.

Ageing is a major risk factor for cancer metastasis but the underlying mechanisms remain unclear. Here, we characterised ageing effects on cancer-induced endothelial-mesenchymal transition (EndMT) in the pulmonary circulation of female BALB/c mice in a metastatic 4T1 breast cancer model. The effect of intravenously injected 4T1 cells on pulmonary endothelium, pulmonary metastasis, lung tissue architecture, and systemic endothelium was compared between 40-week-old and 20-week-old mice. The 40-week-old mice showed features of ongoing EndMT in their lungs before 4T1 breast cancer cell injection. Moreover, they had preexisting endothelial dysfunction in the aorta detected by in vivo magnetic resonance imaging (MRI) compared to 20-week-old mice. The injection of 4T1 breast cancer cells into 40-week-old mice resulted in rapid EndMT progression in their lungs. In contrast, injection of 4T1 breast cancer cells into 20-week-old mice resulted in initiation and less pronounced EndMT progression. Although the number of metastases did not differ significantly between 20-week-old and 40-week-old mice, the lungs of older mice displayed altered lung tissue architecture and biochemical content, reflected in higher Amide II/Amide I ratio, higher fibronectin levels, and hypoxia-inducible factor 1 subunit alpha (HIF1α) levels as well as lower nitric oxide (NO) production. Our results indicate that age-dependent pre-existing endothelial dysfunction in the pulmonary endothelium of 40-week-old mice predisposed them to rapid EndMT progression in the presence of circulating 4T1 breast cancer cells what might contribute to a more severe metastatic breast cancer phenotype in these ageing mice compared to younger mice.

Myeloid cell reprogramming alleviates immunosuppression and promotes clearance of metastatic lesions.

Suppressive myeloid cells, including monocyte and neutrophil populations, play a vital role in the metastatic cascade and can inhibit the anti-tumor function of cytotoxic T-cells. Cargo-free polymeric nanoparticles (NPs) have been shown to modulate innate immune cell responses in multiple pathologies of aberrant inflammation. Here, we test the hypothesis that the intravenous administration of drug-free NPs in the 4T1 murine model of metastatic triple-negative breast cancer can reduce metastatic colonization of the lungs, the primary metastatic site, by targeting the pro-tumor immune cell mediators of metastatic progression. In vivo studies demonstrated that NP administration reprograms the immune milieu of the lungs and reduces pulmonary metastases. Single-cell RNA sequencing of the lungs revealed that intravenous NP administration alters myeloid cell phenotype and function, skewing populations toward inflammatory, anti-tumor phenotypes and away from pro-tumor phenotypes. Monocytes, neutrophils, and dendritic cells in the lungs of NP-treated mice upregulate gene pathways associated with IFN signaling, TNF signaling, and antigen presentation. In a T-cell deficient model, NP administration failed to abrogate pulmonary metastases, implicating the vital role of T-cells in the NP-mediated reduction of metastases. NPs delivered as an adjuvant therapy, following surgical resection of the primary tumor, led to clearance of established pulmonary metastases in all treated mice. Collectively, these results demonstrate that the in vivo administration of cargo-free NPs reprograms myeloid cell responses at the lungs and promotes the clearance of pulmonary metastases in a method of action dependent on functional T-cells.

Multiparametric MRI enables for differentiation of different degrees of malignancy in two murine models of breast cancer.

The objective of this study was to non-invasively differentiate the degree of malignancy in two murine breast cancer models based on identification of distinct tissue characteristics in a metastatic and non-metastatic tumor model using a multiparametric Magnetic Resonance Imaging (MRI) approach. The highly metastatic 4T1 breast cancer model was compared to the non-metastatic 67NR model. Imaging was conducted on a 9.4 T small animal MRI. The protocol was used to characterize tumors regarding their structural composition, including heterogeneity, intratumoral edema and hemorrhage, as well as endothelial permeability using apparent diffusion coefficient (ADC), T1/T2 mapping and dynamic contrast-enhanced (DCE) imaging. Mice were assessed on either day three, six or nine, with an i.v. injection of the albumin-binding contrast agent gadofosveset. Ex vivo validation of the results was performed with laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), histology, immunhistochemistry and electron microscopy. Significant differences in tumor composition were observed over time and between 4T1 and 67NR tumors. 4T1 tumors showed distorted blood vessels with a thin endothelial layer, resulting in a slower increase in signal intensity after injection of the contrast agent. Higher permeability was further reflected in higher Ktrans values, with consecutive retention of gadolinium in the tumor interstitium visible in MRI. 67NR tumors exhibited blood vessels with a thicker and more intact endothelial layer, resulting in higher peak enhancement, as well as higher maximum slope and area under the curve, but also a visible wash-out of the contrast agent and thus lower Ktrans values. A decreasing accumulation of gadolinium during tumor progression was also visible in both models in LA-ICP-MS. Tissue composition of 4T1 tumors was more heterogeneous, with intratumoral hemorrhage and necrosis and corresponding higher T1 and T2 relaxation times, while 67NR tumors mainly consisted of densely packed tumor cells. Histogram analysis of ADC showed higher values of mean ADC, histogram kurtosis, range and the 90th percentile (p90), as markers for the heterogenous structural composition of 4T1 tumors. Principal component analysis (PCA) discriminated well between the two tumor models. Multiparametric MRI as presented in this study enables for the estimation of malignant potential in the two studied tumor models via the assessment of certain tumor features over time.

Immune State Conversion of the Mesenteric Lymph Node in a Mouse Breast Cancer Model.

Secondary lymphoid tissues, such as the spleen and lymph nodes (LNs), contribute to breast cancer development and metastasis in both anti- and pro-tumoral directions. Although secondary lymphoid tissues have been extensively studied, very little is known about the immune conversion in mesenteric LNs (mLNs) during breast cancer development. Here, we demonstrate inflammatory immune conversion of mLNs in a metastatic 4T1 breast cancer model. Splenic T cells were significantly decreased and continuously suppressed IFN-γ production during tumor development, while myeloid-derived suppressor cells (MDSCs) were dramatically enriched. However, T cell numbers in the mLN did not decrease, and the MDSCs only moderately increased. T cells in the mLN exhibited conversion from a pro-inflammatory state with high IFN-γ expression to an anti-inflammatory state with high expression of IL-4 and IL-10 in early- to late-stages of breast cancer development. Interestingly, increased migration of CD103+CD11b+ dendritic cells (DCs) into the mLN, along with increased (1→3)-β-D-glucan levels in serum, was observed even in late-stage breast cancer. This suggests that CD103+CD11b+ DCs could prime cancer-reactive T cells. Together, the data indicate that the mLN is an important lymphoid tissue contributing to breast cancer development.

Engineered Hybrid Treg-Targeted Nanosomes Restrain Lung Immunosuppression by Inducing Intratumoral CD8+T Cell Immunity

IntroductionTumor immunotherapy is a key therapeutic paradigm for the treatment of several malignancies. However, in metastatic lung cancer, classical immunotherapy regimes are ineffective due to regulatory T cell (Treg)-related immunosuppression and tumor relapse.MaterialsTo address this issue, we designed specific biocompatible Treg-targeted nanocarriers (NCs) as a model of immune-based nanotherapy, in order to target Treg-related immunosuppression in the lung tumor microenvironment. This is achieved through the combination of Dasatinib and Epacadostat integrated into biodegradable nanosomes which can inhibit and reverse Treg-supporting immunosuppression. Flow cytometry and immunofluorescence analysis, PET/CT scan, PTT/PA imaging and the Balb/c tumor model were used to explore the anti-tumor effect of Treg-targeted NCs both in vitro and in vivo.ResultsFindings reveal that NC treatment triggered substantial tumor cell apoptosis and drastically decreased tumor volume followed by downregulation of Ki-67 antigen expression, respectively. Drug circulation time was also increased as shown by biodistribution analysis accompanied by greater accumulation in lung and peripheral tissues. Intratumoral Th1 cytokines’ expression was also increased, especially TNF-A, IL-12 by 42%, and IL-6 by 18% compared to PBS treatment. In addition, the presence of mature CD80+/CD86+dendritic cells (DCs) revealed T cell enrichment and a decline in MDSC infiltration and myeloid subsets. Interestingly, a significant decline of Gr/CD11b myeloid cell population in blood and tissue samples was also observed. This immune activation can be attributed to the enhanced PTT efficiency and tumor targeting ability of the nanospheres which under near infrared (NIR) exposure can prompt highly efficient tumor ablation. We also demonstrated their therapeutic efficacy against 4T1 metastatic breast cancer model. Additionally, the photothermal therapy in combination with PD-L1 checkpoint blockade therapy exerted long-term tumor control over both primary and distant tumors.DiscussionOverall, our findings present a novel nano-enabled platform for the inhibition of Treg-dependent immunosuppression in NSCLC and provide a novel nanotherapeutic strategy for the treatment of metastatic neoplasia.