Triple-negative Breast Cancer Lung Research Articles

Abstract 1275: In vivo functional CRISPR screens identifies metabolic dependencies mediating triple negative breast cancer lung metastasis

Abstract Metastasis is responsible for about 90% of cancer-related mortality. Throughout the metastatic cascade, cancer cells requires substantial metabolic rewiring to adapt the nutrient micro-environmental changes enabling dissemination and seeding in distant organs. Recent insights into importance of the tumor-intrinsic metabolic reprograming has been implicated in immune escape, yet altered immune responses imposed by tumor metabolism during metastasis are poorly understood. Here, we generated genetic perturbations in triple-negative breast cancer cells using metabolism-focused CRISPR knockout library and performed in vivo screens in both immunocompetent and immunodeficient mice. By comparing enriched sgRNAs in lung metastases or primary tumors, we identify loss of one gene related to long chain fatty acid (LCFA) β-oxidation (FAO) potentiates metastatic dissemination in immunocompetent mice but no in immunodeficient mice, suggesting that impaired FAO in tumor cells promotes breast cancer lung metastasis through an lymphocytes-dependent manner. We confirmed individual sgRNAs targeting this FAO gene from the primary screen dramatically accelerate metastasis in different breast cancer orthotopic models. Specifically, we find that low expression of this gene in primary tumors of advanced breast cancer patients with high grade tumors and positive lymph nodes (LN+) is associated with reduced overall survival. In addition, deletion of this gene generates an immunosuppressive microenvironment in the lung metastatic tumors with increased neutrophils, monocytes and decreased TNFα+-IFNγ+ CD8+ T cells. Mechanistically, impaired FAO in tumor cells causes mitochondria imbalance under lipid metabolism stress, consequently triggers inflammatory response on establishing a neutrophil-rich metastatic niche via mitochondrial nucleic acid-driven innate immunity. Altogether, our data provides a new insight on how tumor cell lipid metabolism impacting immune interplay during metastatic progression, nominating potential anti-tumor immunotherapies. Citation Format: Xiaoyong Wang, Shu-ting Chou, Verra Ngwa, Yoonha Hwang, Breelyn Karno, Deanna Edwards, Jin Chen. In vivo functional CRISPR screens identifies metabolic dependencies mediating triple negative breast cancer lung metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1275.

Boosting the Potential of Chemotherapy in Advanced Breast Cancer Lung Metastasis via Micro‐Combinatorial Hydrogel Particles (Adv. Sci. 10/2023)

Drug Delivery The cover shows multiple metastatic nodules developing at different locations within the lungs. Deformable micro-combinatorial hydrogel particles (μCGP) are transported by the blood flow, accumulate proximally to the nodules, and release in a sustained fashion thereof the therapeutic cargo—docetaxel nanomedicines. In an advanced triple negative breast cancer lung metastasis model, μCGP amplify the chemotherapy potential and boost animal survival beyond 5 months. More details can be found in article number 2205223 by Paolo Decuzzi and co-workers.

Role of AMPK and Akt in triple negative breast cancer lung colonization

Triple negative breast cancer (TNBC) is an aggressive disease with a 5-y relative survival rate of 11% after distant metastasis. To survive the metastatic cascade, tumor cells remodel their signaling pathways by regulating energy production and upregulating survival pathways. AMP-activated protein kinase (AMPK) and Akt regulate energy homeostasis and survival, however, the individual or synergistic role of AMPK and Akt isoforms during lung colonization by TNBC cells is unknown. The purpose of this study was to establish whether targeting Akt, AMPKα or both Akt and AMPKα isoforms in circulating cancer cells can suppress TNBC lung colonization. Transient silencing of Akt1 or Akt2 dramatically decreased metastatic colonization of lungs by inducing apoptosis or inhibiting invasion, respectively. Importantly, transient pharmacologic inhibition of Akt activity with MK-2206 or AZD5363 inhibitors did not prevent colonization of lung tissue by TNBC cells. Knockdown of AMPKα1, AMPKα2, or AMPKα1/2 also had no effect on metastatic colonization of lungs. Taken together, these findings demonstrate that transient decrease in AMPK isoforms expression alone or in combination with Akt1 in circulating tumor cells does not synergistically reduce TNBC metastatic lung colonization. Our results also provide evidence that Akt1 and Akt2 expression serve as a bottleneck that can challenge colonization of lungs by TNBC cells.

Normalizing the Microenvironment Overcomes Vessel Compression and Resistance to Nano-immunotherapy in Breast Cancer Lung Metastasis.

Nano‐immunotherapy regimens have high potential to improve patient outcomes, as already demonstrated in advanced triple negative breast cancer with nanoparticle albumin‐bound paclitaxel and the immune checkpoint blocker (ICB) atezolizumab. This regimen, however, does not lead to cures with median survival lasting less than two years. Thus, understanding the mechanisms of resistance to and development of strategies to enhance nano‐immunotherapy in breast cancer are urgently needed. Here, in human tissue it is shown that blood vessels in breast cancer lung metastases are compressed leading to hypoxia. This pathophysiology exists in murine spontaneous models of triple negative breast cancer lung metastases, along with low levels of perfusion. Because this pathophysiology is consistent with elevated levels of solid stress, the mechanotherapeutic tranilast, which decompressed lung metastasis vessels, is administered to mice bearing metastases, thereby restoring perfusion and alleviating hypoxia. As a result, the nanomedicine Doxil causes cytotoxic effects into metastases more efficiently, stimulating anti‐tumor immunity. Indeed, when combining tranilast with Doxil and ICBs, synergistic effects on efficacy, with all mice cured in one of the two ICB‐insensitive tumor models investigated is resulted. These results suggest that strategies to treat breast cancer with nano‐immunotherapy should also include a mechanotherapeutic to decompress vessels.

Abstract 3074: Targeting sanctuary sites of cancer: Novel approaches to treatment of lung localized tumors

Abstract The lung is one of the most common sites of cancer metastasis. We hypothesize that the lack of effective chemotherapeutics for lung localized tumors might in part be due to the lungs acting as a sanctuary site for cancer cells. To identify more effective anticancer therapies, we focused on the discovery and development of drugs reaching tumor sanctuaries either by design and synthesis of novel drugs, use of a compatible delivery system or a combination of both. To test our hypothesis and develop a novel approach to eliminate cancer sanctuary sites we focused on anthracycline antibiotics, specifically on the therapeutic limitations of doxorubicin (DOX). We have developed Annamycin (ANN), a novel, non-cardiotoxic DOX analog formulated in multilamellar liposomes (L-ANN) with high activity against multidrug resistant (MDR) cancer cells and an organ distribution dramatically different from that of DOX. L-ANN is currently in multicenter Phase I clinicals studies. Objective: The objective of our study was to confirm high uptake and retention of free ANN in lungs, demonstrate the same for L-ANN, and to assess the efficacy of L-ANN in lung metastatic cancer models. Methods: Pharmacokinetics and biodistribution of ANN were tested using LC/MS/MS in naïve CD-1 mice after intravenous administration of the drug. In vitro activity of ANN was analyzed in different human and murine cancer cells. Cellular uptake and subcellular distribution of ANN was assessed using fluorescent microscopy and FACS. Efficacy of L-ANN was tested in CT26 colon and 4T1 triple negative breast cancer lung metastatic mouse models. Results: Biodistribution experiments showed high levels of ANN in lung tissue either for free or L-ANN that exceeded that in plasma by 7 to 10 fold. ANN levels in lungs were also more than 6-fold greater than that of DOX. In addition, on a cellular level, immunofluorescence and FACS analysis demonstrated significantly higher uptake of ANN than that of DOX, as well as potent induction of double-strand DNA breaks. ANN showed consistently high cytotoxicity across all tested cell lines including MDR cells with IC50s in the low nanomolar range. Based on bioluminescent imaging in models of lung localized cancers, L-ANN displayed high antitumor activity ranging from inhibition of tumor progression to complete tumor eradication. Importantly, the delay in tumor growth and, in some cases, tumor regression led to significant improvement of survival in all tested models. Conclusion: The high cytotoxic potency of ANN against MDR cancer cells is related, in part, to its ability to overcome ABC transporter-mediated efflux and, in contrast to DOX, achieve high intracellular uptake. A greatly increased concentration of ANN in the lung, as compared to DOX, leads to high drug efficacy in vivo in lung-localized tumor models. In summary, our studies (1) support the hypothesis of lungs being a sanctuary site for cancer cells and (2) demonstrate that effective targeting of cancers metastatic to the lung is possible by chemical modification of clinically used but currently ineffective drugs, especially in combination with appropriate drug delivery. In more general terms, these studies indicate that the proposed approach can also lead to the identification and elimination of cancer sanctuary sites other than the lungs and creation of more effective anticancer therapies. Citation Format: Rafal Zielinski, Krzysztol Grela, Roberto Cardena Zuniga, Yaan Kang, Stanislaw Skora, Edward Felix, Julia Tuchalska-Czuron, Izabela Fokt, Waldemar Priebe. Targeting sanctuary sites of cancer: Novel approaches to treatment of lung localized tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3074.

Multimodal Fluorescence and Bioluminescence Imaging Reveals Transfection Potential of Intratracheally Administered Polyplexes for Breast Cancer Lung Metastases.

Local delivery of anticancer agents or gene therapeutics to lung tumors can circumvent side effects or accumulation in non-target organs, but accessibility via the alveolar side of the blood-air barrier remains challenging. Polyplexes based on plasmid and linear polyethylenimine (LPEI) transfect healthy lung tissue when applied intravenously (i.v.) in the mouse, but direct delivery into the lungs results in low transfection of lung tissue. Nevertheless, LPEI could offer the potential to transfect lung tumors selectively, if accessible from the alveolar side. This study combined near infrared fluorescent protein 720 (iRFP720) and firefly luciferase as reporter genes for detection of tumor lesions and transfection efficiency of LPEI polyplexes, after intratracheal microspraying in mice bearing 4T1 triple negative breast cancer lung metastases. Simultaneous flow cytometric analysis of iRFP720 and enhanced green fluorescent protein expression in vitro demonstrated the potential to combine these reporter genes within transfection studies. Polyplex biophysics was characterized by single nanoparticle tracking analysis (NTA) to monitor physical integrity after microspraying in vitro. 4T1 cells were transduced with iRFP720-encoding lentivirus and evaluated by flow cytometry for stable iRFP720 expression. Growth of 4T1-iRFP720 cells was monitored in Balb/c mice by tomographic near infrared imaging, tissue and tumor morphology by computed tomography and magnetic resonance imaging. In 4T1-iRFP720 tumor-bearing mice, intratracheal administration of luciferase-encoding plasmid DNA by LPEI polyplexes resulted in successful tumor transfection, as revealed by bioluminescence imaging.

Abstract 3869: MicroRNA-200b blocks breast cancer lung metastasis in an orthotopic mouse mammary xenograft tumor model.

Abstract Triple negative, or basal-like, breast cancer is a highly invasive and metastatic form of breast cancer, and is difficult to treat due to the lack of target therapies. Epithelial to mesenchymal transition (EMT) refers to a developmental program that converts epithelial cells to mesenchymal cells, therefore enabling cells to migrate and invade. EMT is tightly controlled during development, and recent studies showed that EMT is often reactivated in cancer and plays an important role in cancer invasion and metastasis, and that EMT can also be regulated by microRNAs. MicroRNAs (miRs) are a large family of small non-coding RNAs that negatively regulate protein-coding gene expression posttranscriptionally. Although the miR-200 family members have been shown to be able to inhibit EMT, their effects on cancer metastasis continue to be controversial. We aim to investigate the effect of miR-200b on triple negative breast cancer lung metastasis and the underlying mechanism. It was found that stably expressing miR-200b in MDA-MB-231 cells blocked lung metastasis in an orthotopic mouse mammary xenograft tumor model. In vitro studies showed that stably expressing miR-200b significantly reduced triple negative breast cancer cell proliferation, migration, and invasion. Furthermore, stably expressing miR-200b also decreased tumor growth and angiogenesis in the orthotopic mouse mammary xenograft tumors. These findings suggest that miR-200b blocks breast cancer lung metastasis through its strong inhibitory effect on multiple-steps including proliferation, migration, invasion and angiogenesis. Citation Format: Brock Humphries, Zhishan Wang, Chengfeng Yang. MicroRNA-200b blocks breast cancer lung metastasis in an orthotopic mouse mammary xenograft tumor model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3869. doi:10.1158/1538-7445.AM2013-3869

Abstract P6-13-02: Overcoming therapy resistance of metastatic breast cancer by enhanced tumor delivery of polymeric doxorubicin

Abstract Metastatic breast cancers are treated with chemotherapy drugs in clinic. However, patients usually develop therapy resistance quickly due to intrinsic and acquired mechanisms. Poor drug delivery to tumor cells may also contribute to therapy resistance. To address both biological and mass transport barriers to effective therapy, we developed a new doxorubicin-based formulation, polymeric doxorubicin (pDox), to be delivered using a porous silicon drug carrier, the multistage vector (MSV). We show the novel, rationally designed MSV/pDox drug enriches in tumor tissues and has considerable therapeutic efficacy in two animal models of triple negative breast cancer lung metastasis, conferring a significant survival advantage to animals treated with MSV/pDox over all other control groups (80% survival for the MSV/pDox group at 24 weeks post treatment vs. 0% survival for all others, log-rank test, p < 0.001). Mechanistically, pDox is released from the MSV as nanoparticles by controlled, sustained kinetics, and enters tumor cells by vesicular transport. The active doxorubicin is released in the acidic environment in lysosomes, exits vesicles in the perinuclear region, and enters the nucleus for drug action. This effective mass transport mechanism kills both the bulk and therapy resistant cells, but avoids cardiac damage. As a result, this new nanodrug has a significantly higher therapeutic window over doxorubicin and Doxil. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-13-02.