Lung Micrometastases Research Articles

In Vivo PET Detection of Lung Micrometastasis in Mice by Targeting Endothelial VCAM-1 Using a Dual-Contrast PET/MRI Probe.

Current clinical diagnostic imaging methods for lung metastases are sensitive only to large tumours (1-2 mm cross-sectional diameter), and early detection can dramatically improve treatment. We have previously demonstrated that an antibody-targeted MRI contrast agent based on microparticles of iron oxide (MPIO; 1 μm diameter) enables the imaging of endothelial vascular cell adhesion molecule-1 (VCAM-1). Using a mouse model of lung metastasis, upregulation of endothelial VCAM-1 expression was demonstrated in micrometastasis-associated vessels but not in normal lung tissue, and binding of VCAM-MPIO to these vessels was evident histologically. Owing to the lack of proton MRI signals in the lungs, we modified the VCAM-MPIO to include zirconium-89 (89Zr, t1/2 = 78.4 h) in order to allow the in vivo detection of lung metastases by positron emission tomography (PET). Using this new agent (89Zr-DFO-VCAM-MPIO), it was possible to detect the presence of micrometastases within the lung in vivo from ca. 140 μm in diameter. Histological analysis combined with autoradiography confirmed the specific binding of the agent to the VCAM-1 expressing vasculature at the sites of pulmonary micrometastases. By retaining the original VCAM-MPIO as the basis for this new molecular contrast agent, we have created a dual-modality (PET/MRI) agent for the concurrent detection of lung and brain micrometastases.

Exosomal HMGB1 educate PD-L1+-tumor associated macrophages via glycometabolic reprogramming to induce lung-tropic metastasis of triple negative breast cancer.

1090 Background: Lung metastasis occurs in one third of triple negative breast cancer (TNBC), the underlying molecular mechanism remains largely unclear. Mounting evidence suggests that PD-L1+-tumor associated macrophages (PD-L1+-TAMs) are closely associated with immune suppression, invasion, and metastasis. Tumor-derived exosomes have been reported to remodel tumor microenvironment and accelerate metastasis through packing and delivering a variety of biologically active molecules. The aim of this study was to investigate whether TNBC-derived exosomes were able to polarize lung macrophages towards PD-L1+-TAMs and promote a pre-metastatic immunosuppressive niche, therefore discovering the underlying mechanism of lung-tropic metastasis. Methods: Exosomes obtained from EO771 TNBC cell line (EO771/exo) and HC11 normal breast epithelial cell line (HC11/exo) were characterized. Absorption of exosomes by macrophages was observed by confocal microscopy. Mass spectrometry was used to identify the significantly highly expressed HMGB1 protein in EO771/exo, and ligands of HMGB1 on lung macrophages were detected by co-immunoprecipitation. Significance of EO771/exo and HMGB1 in inducing PD-L1+-TAMs and generating a pre-metastatic immunosuppressive niche were confirmed by both in vitro and in vivo studies. Functions of EO771/exo and HMGB1 on glycolysis and lactate production were evaluated by metabolism assays. Results: EO771/exo could polarize macrophages toward PD-L1+-TAMs and generate a pre-metastatic immunosuppressive niche. Mass spectrometry, clinical samples, and bioinformatics analysis showed that HMGB1 was significantly expressed in EO771/exo and was involved in cellular communication and metabolism processes. Co-immunoprecipitation and 3-dimensional modeling suggested a strong binding of HMGB1 with Tim-3 ligand on lung macrophages. Macrophages incubated with EO771/exo exhibited an enhanced glycolysis and lactate production. Flow cytometry demonstrated that green fluorescent protein (GFP)-EO771 tumor-bearing mice had more lung micro-metastases than brain and liver. Immunofluorescence and laser confocal microscopy demonstrated that EO771/exo were able to increase lung micro-metastases and escalate PD-L1 expression on lung macrophages. Moreover, elevated PD-L1+-TAMs and reduced CD3+CD8+ T lymphocytes were detected in TNBC positive axillary lymph nodes and lung metastasis specimens than TNBC negative axillary lymph nodes. Conclusions: Exosomal HMGB1 from TNBC could target Tim-3 ligand on lung macrophages and accelerate glycolysis and lactate production to induce PD-L1+-TAMs, thereby suppressing CD3+CD8+ T lymphocytes immunity and generating a pre-metastasis immune-suppressive niche. Our research would uncover a novel mechanism of lung-tropic metastasis.

Understanding clonal heterogeneity and mechanisms of methotrexate resistance in two osteosarcoma PDX models.

e23512 Background: Osteosarcoma is a primary malignant bone tumor that is known to have significant intratumoral heterogeneity with branched cancer evolution. The lung is the most frequent site of metastatic disease and relapse occurs in more than 30 % children and young adults. It has been revealed that the metastatic ability and resistance of cancers including osteosarcoma might be due to the presence of a subset of cells within the intra-tumorally heterogeneous population. We investigated the emergence of resistant clonal subpopulations following targeted methotrexate treatment in two patient derived xenograft (PDX) models in vitro and in vivo. Our aim was to not only track the subclonal population of cells that establish in the lungs but also to identify clonal drivers and the mutational signature of the genes and pathways involved in MTX resistance. Methods: We previously established 20 barcoded osteosarcoma PDX models and demonstrated that the application of methotrexate (MTX-IC95) treatment as a selective pressure in seven models produces clonal subpopulations that were more resistant to MTX. In the current work we used barcoded next generation and RNA sequencing techniques to map the clonal and transcriptomic architecture of MTX resistant single clones in M36 and OS252 PDX models. We next injected the barcoded PDX cells intratibially in twenty severe combined immunodeficiency disease (SCID) mice to track the clonal population/subpopulation of cells that metastasize to the lungs. Following primary tumor growth, limb amputation was carried out and mice were treated with methotrexate or vehicle for two weeks. Primary tumor, blood and lung samples were collected after death from metastasis or euthanasia. All samples collected were snap frozen in liquid nitrogen and stored at -800C. DNA and RNA was extracted and analyzed by PCR, NGS or WES to map clonality, mutational and evolutionary profiles. Results: MTX treated single clones showed diversity and barcode abundance when compared to vehicle; barcode abundance reduced in the resistant clonal subpopulations when compared to the vehicles. Ten unique subclones were shared in both vehicle and MTX resistant clones in M36 while OS252 had only one shared subclone respectively. More than 50% of the injected mice developed primary tumors and matched lung micrometastases. We are currently analyzing both targeted and RNA sequencing results to further understand clonality, the mutational signature of the genes (DHFR, MTR, FPGS, GGH) and pathways involved in MTX resistance. Conclusions: We investigated mechanism of methotrexate resistance in two barcoded PDX models in vitro and in vivo using cytotoxicity, next generation and RNA sequencing techniques. Targeted amplicon sequencing of key genes involved in antifolate pathway will yield new information on clonal evolution and mechanisms of resistance.

Abstract PO1-06-11: Exosomal HMGB1 induce PD-L1+-tumor associated macrophages through glycometabolic reprogramming to promote lung-tropic metastasis of triple negative breast cancer

Abstract Background: Lung metastasis occurs in 30% of triple negative breast cancer (TNBC), the detailed molecular mechanism remains largely unexplored. Accumulating evidence suggests that PD-L1+-tumor associated macrophages (PD-L1+-TAMs) are closely related to immune suppression, invasion, and metastasis. Tumor-derived exosomes have been reported to remodel tumor microenvironment and accelerate metastasis via packing and delivering a variety of biologically active molecules. Our previous studies found that exosomes from breast cancer cells polarized macrophages toward TAMs within the primary tumor nest. The aim of this study was to reveal whether TNBC-derived exosomes were able to polarize lung macrophages towards PD-L1+-TAMs and generate a pre-metastatic immunosuppressive niche, therefore discovering the underlying mechanism of lung-tropic metastasis. Methods: Exosomes isolated from EO771 TNBC cell line (EO771/exo) and HC11 normal breast epithelial cell line (HC11/exo) were characterized. Uptake of exosomes by macrophages was visualized by confocal microscopy. Mass spectrometry was applied to identify the significantly highly expressed HMGB1 protein in EO771/exo, and ligands of HMGB1 on lung macrophages were detected by co-immunoprecipitation. Functions of EO771/exo and HMGB1 in inducing PD-L1+-TAMs and generating a pre-metastatic immunosuppressive niche were confirmed by both in vitro and in vivo studies. Roles of EO771/exo and HMGB1 on glycolysis and lactate production were evaluated by metabolism assays. Associations between PD-L1+-TAMs and CD3+CD8+ T lymphocytes within TNBC axillary lymph nodes and lung metastasis specimens were analyzed by immunofluorescence and immunohistochemistry. Results: Confocal microscopy showed constant internalization and absorption of EO771/exo by macrophages. EO771/exo could polarize macrophages toward PD-L1+-TAMs and generate a pre-metastatic immunosuppressive niche, with respect to HC11/exo. Mass spectrometry, clinical samples, and bioinformatics analysis revealed that HMGB1 was highly expressed in EO771/exo and was involved in cellular communication and metabolism processes. Co-immunoprecipitation and 3-dimensional modeling indicated a strong binding of HMGB1 with Tim-3 ligand on lung macrophages. Macrophages treated with EO771/exo displayed an enhanced glycolysis and lactate production. Flow cytometry demonstrated that green fluorescent protein (GFP)-EO771 tumor-bearing mice had more lung micro-metastases than brain and liver. Immunofluorescence and laser confocal microscopy confirmed that EO771/exo, with respect to HC11/exo, were able to increase lung micro-metastases and escalate PD-L1 expression on lung macrophages. Moreover, elevated PD-L1+-TAMs and reduced CD3+CD8+ T lymphocytes were found in TNBC positive axillary lymph nodes and lung metastasis specimens than TNBC negative axillary lymph nodes. Conclusions: Exosomal HMGB1 from TNBC could target Tim-3 ligand on lung macrophages and accelerate glycolysis and lactate production to induce PD-L1+-TAMs, thereby suppressing CD3+CD8+ T lymphocytes immunity and generating a pre-metastasis immune-suppressive niche. Our research would uncover a novel mechanism of lung-tropic metastasis and provide a new therapeutic approach for TNBC treatment. Keywords: breast cancer; lung metastasis; exosomes; tumor-associated macrophages; metabolic reprogramming Citation Format: Wei-Xian Chen, Jia-Hao Wu, Tian-Cheng Cheng, Sujin Yang. Exosomal HMGB1 induce PD-L1+-tumor associated macrophages through glycometabolic reprogramming to promote lung-tropic metastasis of triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-06-11.

The incorporation of acetylated LAP-TGF-β1 proteins into exosomes promotes TNBC cell dissemination in lung micro-metastasis

Triple-negative breast cancer (TNBC) stands as the breast cancer subtype with the highest recurrence and mortality rates, with the lungs being the common site of metastasis. The pulmonary microenvironment plays a pivotal role in the colonization of disseminated tumor cells. Herein, this study highlights the crucial role of exosomal LAP-TGF-β1, the principal form of exosomal TGF-β1, in reshaping the pulmonary vascular niche, thereby facilitating TNBC lung metastasis. Although various strategies have been developed to block TGF-β signaling and have advanced clinically, their significant side effects have limited their therapeutic application. This study demonstrates that in lung metastatic sites, LAP-TGF-β1 within exosomes can remarkably reconfigure the pulmonary vascular niche at lower doses, bolstering the extravasation and colonization of TNBC cells in the lungs. Mechanistically, under the aegis of the acetyltransferase TIP60, a non-canonical KFERQ-like sequence in LAP-TGF-β1 undergoes acetylation at the K304 site, promoting its interaction with HSP90A and subsequent transport into exosomes. Concurrent inhibition of both HSP90A and TIP60 significantly diminishes the exosomal burden of LAP-TGF-β1, presenting a promising therapeutic avenue for TNBC lung metastasis. This study not only offers fresh insights into the molecular underpinnings of TNBC lung metastasis but also lays a foundation for innovative therapeutic strategies.

GK-1 effectively reduces angiogenesis and prevents T cell exhaustion in a breast cancer murine experimental model

Breast cancer is the leading malignancy in women worldwide, both in terms of incidence and mortality. Triple-negative breast cancer (TNBC) is the type with the worst clinical outcomes and with fewer therapeutic options than other types of breast cancer. GK-1 is a peptide that in the experimental model of the metastatic 4T1 breast cancer has demonstrated anti-tumor and anti-metastatic properties. Herein, GK-1 (5 mg/kg, i.v.) weekly administrated not only decreases tumor growth and the number of lung macro-metastases but also lung and lymph nodes micro-metastases. Histological analysis reveals that GK-1 reduced 57% of the intra-tumor vascular areas, diminished the leukemoid reaction's progression, and the spleens' weight and length. A significant reduction in VEGF-C, SDF-1, angiopoietin-2, and endothelin-1 angiogenic factors was induced. Moreover, GK-1 prevents T cell exhaustion in the tumor-infiltrating lymphocytes (TILs) decreasing PD-1 expression. It also increased IFN-γ and granzyme-B expression and the cytotoxic activity of CD8+ TILs cells against tumor cells. All these features were found to be associated with a better antitumor response and prognosis. Altogether, these results reinforce the potential of GK-1 to improve the clinical outcome of triple-negative breast cancer immunotherapy. Translation research is ongoing towards its evaluation in humans.

Synthesis and Evaluation of 177Lu-DOTA-PD-L1-i and 225Ac-HEHA-PD-L1-i as Potential Radiopharmaceuticals for Tumor Microenvironment-Targeted Radiotherapy.

Current cancer therapies focus on reducing immunosuppression and remodeling the tumor microenvironment to inhibit metastasis, cancer progression, and therapeutic resistance. Programmed death receptor 1 (PD-1) is expressed on immune T cells and is one of the so-called checkpoint proteins that can suppress or stop the immune response. To evade the immune system, cancer cells overexpress a PD-1 inhibitor protein (PD-L1), which binds to the surface of T cells to activate signaling pathways that induce immune suppression. This research aimed to synthesize PD-L1 inhibitory peptides (PD-L1-i) labeled with lutetium-177 (177Lu-DOTA-PD-L1-i) and actinium-225 (225Ac-HEHA-PD-L1-i) and to preclinically evaluate their potential as radiopharmaceuticals for targeted radiotherapy at the tumor microenvironment level. Using PD-L1-i peptide as starting material, conjugation with HEHA-benzene-SCN and DOTA-benzene-SCN was performed to yield DOTA-PD-L1-i and HEHA-PD-L1-I, which were characterized by FT-IR, UV-vis spectroscopy, and HPLC. After labeling the conjugates with 225Ac and 177Lu, cellular uptake in HCC827 cancer cells (PD-L1 positive), conjugate specificity evaluation by immunofluorescence, radiotracer effect on cell viability, biodistribution, biokinetics, and assessment of radiation absorbed dose in mice with in duced lung micrometastases were performed. 225Ac-HEHA-PD-L1-i and 177Lu-DOTA-PD-L1-i, obtained with radiochemical purities of 95 ± 3% and 98.5 ± 0.5%, respectively, showed in vitro and in vivo specific recognition for the PD-L1 protein in lung cancer cells and high uptake in HCC287 lung micrometastases (>30% ID). The biokinetic profiles of 177Lu-DOTA-PD-L1-i and 225Ac-DOTA-PD-L1-i showed rapid blood clearance with renal and hepatobiliary elimination and no accumulation in normal tissues. 225Ac-DOTA-PD-L1-i produced a radiation dose of 5.15 mGy/MBq to lung micrometastases. In the case of 177Lu-DOTA-PD-L1-i, the radiation dose delivered to the lung micrometastases was ten times (43 mGy/MBq) that delivered to the kidneys (4.20 mGy/MBq) and fifty times that delivered to the liver (0.85 mGy/MBq). Therefore, the radiotherapeutic PD-L1-i ligands of 225Ac and 177Lu developed in this research could be combined with immunotherapy to enhance the therapeutic effect in various types of cancer.

Abstract 2903: Epigenetic modulation of NK cells to improve tumor trafficking and enhance therapeutic efficacy against osteosarcoma

Abstract Osteosarcoma is the most common primary malignant bone tumor. It metastasizes almost exclusively to the lung, and this constitutes the main cause of death in these patients. Immune therapy has shown limited benefit. We investigated whether epigenetic modulation of allogeneic NK cells by the HDAC inhibitor MS-275 will improve tumor trafficking and enhance therapeutic efficacy against osteosarcoma (OS) lung metastases. Human NK cells were purified and expanded from donor buffy coats using recombinant human IL-2 and genetically engineered K562 feeder cells and treated with MS-275 before injection into mice. RNA seq was performed in untreated and MS-275 pre-treated NK cells to determine differentially expressed genes. We also used ChiPseq to determine the histone post-translational modifications associated with the differentially expressed genes induced by the treatment. Culture media from untreated and treated NK cells was collected for cytokine analysis using the Isolight/Isoplexis system. We used our OS-17 human OS experimental lung metastases mouse model and injected untreated or pre-treated NK cells (0.5uM MS-275) IV labeled with near-infrared DiR dye twice a week. Tumor burden was assessed, and NK cells were tracked by Bioluminescence imaging (BLI). Mice were either euthanized 24 hours after the final NK cell treatment for histologic analysis of the lungs or, in a second experiment, analyzed for survival across treatment groups. Some mice from each group were selected for 3D imaging using the Xerra cryofluorescence tomography (CFT) imaging system, allowing for greater resolution of NK cell distribution as compared to the IVIS imaging system. We identified 547 differentially expressed genes between the untreated and MS-275 treated NK cells. Four immune related genes (L1CAM, IFNG, C3 and CD28) were upregulated in the MS-275 treated NK cells and histone H3 and H4 acetylation was found at the identified immunomodulatory genes. Culture media from MS-275 treated NK cells showed significantly increased MIP-1b, IL-6 and IFN-g. Furthermore, there was a significant decrease in lung micro-metastases in the MS-275 pre-treated NK cell group as compared to the untreated control group (199 vs 97 p=0.05). Lastly, Xerra CFT imaging demonstrated greater DiR signal in the lungs of the mice that received MS-275 treated NK cells compared to those that received untreated NK cells, suggesting that MS-275 might lead to greater persistence of NK cells at tumor sites. We conclude that MS-275 has an immunomodulatory effect on NK cells (increase L1CAM, IFNG, C3 and CD28) and increases H3 and H4 acetylation which leads to transcriptional activation of the immune-related genes that contribute to NK cell increased cytolytic function. Pre-treatment of NK cells with MS-275 enhances NK cell therapeutic efficacy and localization and persistence in the lungs. Citation Format: Ariana Rupp Anjier, Nancy Gordon, Shinji Maegawa, Vidya Gopalakrishnan, Juan Bournat, Donghang Cheng, Yanwen Yang, Charles Kingsley, Adam Kulp. Epigenetic modulation of NK cells to improve tumor trafficking and enhance therapeutic efficacy against osteosarcoma [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 2903.

Fibronectin-targeting and metalloproteinase-activatable smart imaging probe for fluorescence imaging and image-guided surgery of breast cancer

Residual lesions in the tumor bed have been a challenge for conventional white-light breast-conserving surgery. Meanwhile, lung micro-metastasis also requires improved detection methods. Intraoperative accurate identification and elimination of microscopic cancer can improve surgery prognosis. In this study, a smart fibronectin-targeting and metalloproteinase-activatable imaging probe CREKA-GK8-QC is developed. CREKA-GK8-QC possesses an average diameter of 21.7 ± 2.5 nm, excellent MMP-9 protein responsiveness and no obvious cytotoxicity. In vivo experiments demonstrate that NIR-I fluorescence imaging of CREKA-GK8-QC precisely detects orthotopic breast cancer and micro-metastatic lesions (nearly 1 mm) of lungs with excellent imaging contrast ratio and spatial resolution. More notably, fluorescence image-guided surgery facilitates complete resection and avoids residual lesions in the tumor bed, improving survival outcomes. We envision that our newly developed imaging probe shows superior capacity for specific and sensitive targeted imaging, as well as providing guidance for accurate surgical resection of breast cancer.Graphical

Sequential targeting of PARP with carboplatin inhibits primary tumour growth and distant metastasis in triple-negative breast cancer

BackgroundPatients with triple-negative breast cancer (TNBC) develop early recurrence. While PARP inhibitors (PARPi) have demonstrated potential in BRCA1/2-mutant (BRCAMUT) TNBC, durable responses will likely be achieved if PARPi are used in combination. It is plausible that sequential administration of a potent PARPi like talazoparib in combination with carboplatin can enhance primary tumour and metastasis inhibition in BRCAMUT and BRCA1/2 wild-type (BRCAWT) TNBCs, and decrease toxicity.MethodsWe evaluated the impact of the concurrent combination of talazoparib and carboplatin on cell survival in 13 TNBC cell lines. We compared the concurrent and sequential combination upon fork replication, migration and invasion. We also used three orthotopic xenograft models to evaluate primary tumour growth, distant metastasis, and toxicity.ResultsConcurrent talazoparib and carboplatin was synergistic in 92.3% of TNBC cell lines, independent of BRCA1/2-mutation status. The sequential combination decreased fork speed in normal cells, but not in TNBC cells. The talazoparib-first sequential combination resulted in a strong reduction in migration (70.4%, P < 0.0001), invasion (56.9%, P < 0.0001), lung micrometastasis (56.4%, P < 0.0001), and less toxicity in a BRCAWT model.ConclusionThe sequential combination of talazoparib and carboplatin is an effective approach to inhibit micrometastatic disease, providing rationale for the use of this combination in early TNBC patients.

A glutathione-activated NIR-II fluorescent probe for precise localization of micrometastases

Accurate detection and localization of micrometastases are extremely important for tumor diagnosis and comprehensive treatment. However, it is clinically difficult to finely localize micrometastases, especially focal lesions smaller than 1 mm. To that purpose, this work designed a tumor microenvironment factor (glutathione, GSH)-activated second near-infrared transparency window (NIR-II) fluorescent probe for visualization of micrometastases. It is constructed by complexing copper ions with 3-mercaptopropionic acid-modified silver sulfide (Ag2S-MPA) quantum dots. The experimental results showed that the probe has good and stable GSH activation properties and can achieve tumor-specific NIR-II fluorescence imaging. Further, in vivo imaging experiments demonstrated that the probe can precisely localize melanomas with a diameter of < 0.1 mm in lung micrometastases. This work provides a potential strategy for the development of novel tools for localization and visualization of micrometastases.

Abstract B008: The role of the androgen receptor in reciprocal activation of breast cancer metastases and the lung epithelium during metastatic outgrowth

Abstract The lung is one of the most common sites of breast cancer (BC) metastasis. A major gap in our ability to effectively treat BC patients with established lung metastases lies in our lack of knowledge regarding how the lung microenvironment is remodeled during metastasis and how this contributes to disease progression. Our data suggest that the lung epithelium, in particular lung type II alveolar epithelial (AT2) cells, contribute to metastatic outgrowth. We hypothesize that BC lung micrometastases activate surrounding lung epithelial cells which, in turn, support the survival and outgrowth of BC metastases within the lung. The overall goal of these studies is to identify factors secreted by resident lung cells that could be used as metastasis-specific therapeutic targets/agents. We utilized immunocompetent mouse metastasis models to study metastatic progression within the lung. Multispectral fluorescent imagining of metastases demonstrated that a wound repair-related phenotype, characterized by chronic inflammation, develops within the lung microenvironment during metastatic outgrowth. We observed an increase in the number and activation of AT2 cells surrounding metastases as they grow. To gain mechanistic insight into how growing metastases may impact AT2 cells, we conducted single-cell RNA-sequencing (scRNAseq) on mouse lungs with high vs. low metastatic burden. This investigation showed that metastatic outgrowth significantly changes AT2 gene expression resulting in a modified secretome. Cell culture experiments, using multiple AT2 cell models, indicated that no contact co-culture with TNBC also caused significant changes in AT2 gene expression. Upstream regulator analysis of these datasets predicted that the androgen receptor (AR) may contribute to AT2 activation during metastatic outgrowth. Compared to non-adjacent tumor-free MMTV-PyMT lung tissue, there was an increased percentage of AR+ cells in the lung adjacent to metastases, which was further associated with large metastases only. Co-immunofluorescence experiments demonstrated that these lung-specific AR+ cells were primarily AT2 cells and fibroblasts, while in human lungs the majority of AR+ cells were AT2 cells and macrophages. Interestingly, co-culturing of TNBC and AT2 cells enhanced BC proliferation, suggesting that following activation, AT2 secreted factors may reciprocally impact metastatic growth. In conclusion, these data indicate that a tumor supportive microenvironment develops as lung metastases grow and suggests that by the time patients present with established/detectable lung metastases, the lung microenvironment has already undergone fundamental alterations that promote metastatic progression. Future studies will elucidate how activated lung cells support metastatic progression and could lead to the development or repurposing of therapeutic strategies to prevent destructive metastatic outgrowth. Citation Format: Jessica L. Christenson, Nicole S. Spoelstra, G. Devon Trahan, Kathleen I. O'Neill, Michelle M. Williams, Jennifer K. Richer. The role of the androgen receptor in reciprocal activation of breast cancer metastases and the lung epithelium during metastatic outgrowth [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B008.

A Genetically Encoded Magnetic Resonance Imaging Reporter Enables Sensitive Detection and Tracking of Spontaneous Metastases in Deep Tissues.

An MRI reporter gene system optimized for tracking metastasis in deep tissues at high resolutions and able to detect spontaneous micrometastases in lungs of mice provides a useful tool for metastasis research.

Osteosarcoma-Derived Exosomes as Potential PET Imaging Nanocarriers for Lung Metastasis.

Lung metastases represent the most adverse clinical factor and rank as the leading cause of osteosarcoma-related death. Nearly 80% of patients present lung micrometastasis at diagnosis not detected with current clinical tools. Herein, an exosome (EX)-based imaging tool is developed for lung micrometastasis by positron emission tomography (PET) using osteosarcoma-derived EXs as natural nanocarriers of the positron-emitter copper-64 (64 Cu). Exosomes are isolated from metastatic osteosarcoma cells and functionalized with the macrocyclic chelator NODAGA for complexation with 64 Cu. Surface functionalization has no effect on the physicochemical properties of EXs, or affinity for donor cells and endows them with favorable pharmacokinetics for in vivo studies. Whole-body PET/magnetic resonance imaging (MRI) images in xenografted models show a specific accumulation of 64 Cu-NODAGA-EXs in metastatic lesions as small as 2-3mm or in a primary tumor, demonstrating the exquisite tropism of EXs for homotypic donor cells. The targetability for lung metastasis is also observed by optical imaging using indocyanine green (ICG)-labeled EXs and D-luciferin-loaded EXs. These findings show that tumor-derived EXs hold great potential as targeted imaging agents for the noninvasive detection of small lung metastasis by PET. This represents a step forward in the biomedical application of EXs in imaging diagnosis with increased translational potential.

Abstract 3126: The secretome of colorectal cancer cell lines cultured in vitro is predictive for their growth and metastatic behavior in an orthotopic in vivo mouse model

Abstract Metastatic disease in solid cancer in general and specifically in colorectal cancer (CRC) is still the main reason for a poor prognosis underlining the need for new therapeutic strategies. Furthermore, a dysregulated secretion is one major driver of cancer progression and invasion and therefore holds promise as a general therapeutic target for the treatment of metastases. The overall aim of this study is to gain a quantitative understanding of the CRC secretome to elucidate the influence of the microenvironment on selected players and vice versa the influence of the secretome in vitro and in vivo. In a first attempt to elucidate the role of secreted factors in colon cancer progression, we cultured 20 CRC cell lines (five PDX derived and 15 commercial lines) in 3D spheroid cultures and compared the secreted cytokine profile with the 2D culture conditions of the same CRC cell line panel. We analyzed 45 cytokines in duplicates by using a human specific magnetic bead-based assay. Based on the cluster analysis of those cytokine profiles we categorized the CRC lines into low metastatic, intermediate and highly metastatic lines. To validate this clustering in vivo we implanted one representative cell line of each cluster orthotopically in immune-compromised NSG mice (n=10 per line in two independent experiments) and followed tumor growth by optical imaging twice a week over the course of up to 50 days. Tumor growth and metastases were confirmed by histological examination and terminal ex vivo imaging of the organs. The murine and human cytokine profile of different organs and the serum of tumor bearing mice was determined as well. The predicted highly metastatic cell line HCT116 displayed a latency of 24 days post implantation: after that time tumor cells were detected via optical imaging not only at the implantation site, but as well in kidney, spleen, liver and lungs in 100% of the examined animals. The intermediate cell line HT29 had a latency of 38 days and showed tumor growth in colon and liver of all animals and lung metastases on 80% of the implanted mice. Mice bearing the PDX derived cell line 269, representing the low metastatic phenotype, had to be sacrificed 50 days post implant. Tumor cells were detected at the implantation site. Micro-metastases in liver and lung were detected in only 75% of the animals. The secretome pattern specific for an individual cell line was predictive for the metastatic potential in vivo in an orthotopic mouse model. Of note, none of the mentioned cell lines metastasized when implanted subcutaneously. The detailed analysis of the mouse and human cytokine profiles will help to elucidate the complex crosstalk between tumor cells and the tumor microenvironment. The modulation of individual signaling cascades in vitro and in vivo will help to gain a deeper understanding of the metastatic process thereby enabling the development of possible new therapies. Citation Format: Jacqueline Bersano, Dorothee Lenhard, Kanstantsin Lashuk, Eva Oswald, Julia Schüler. The secretome of colorectal cancer cell lines cultured in vitro is predictive for their growth and metastatic behavior in an orthotopic in vivo mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3126.

Verteporfin-Loaded Mesoporous Silica Nanoparticles' Topical Applications Inhibit Mouse Melanoma Lymphangiogenesis and Micrometastasis In Vivo.

Photodynamic therapy (PDT) has been pointed out as a candidate for improving melanoma treatment. Nanotechnology application in PDT has increased its efficacy by reducing side effects. Herein, mesoporous silica nanoparticles (MSNs) conjugated with verteporfin (Ver-MSNs), in use with PDT, were administered in mice to evaluate their efficacy on lymphoangiogenesis and micrometastasis in melanoma. Melanoma was induced in mice by the subcutaneous injection of B16-F10 cells. The mice were transcutaneously treated with MSNs, Ver-MSNs, or glycerol and exposed to red light. The treatment was carried out four times until day 20. Lymphangiogenesis and micrometastasis were identified by the immunohistochemical method. Lymphoangiogenesis was halved by MSN treatment compared with the control animals, whereas the Ver-MSN treatment almost abolished it. A similar reduction was also observed in lung micrometastasis. PDT with topically administrated Ver-MSNs reduced melanoma lymphoangiogenesis and lung micrometastasis, as well as tumor mass and angiogenesis, and therefore their use could be an innovative and useful tool in melanoma clinical therapy.

Abstract 1066: Pre-clinical combination of a PARP inhibitor, talazoparib, and carboplatin in triple-negative breast cancer

Abstract Background: PARP inhibitors, such as talazoparib, demonstrated an improvement in progression-free survival as monotherapy in germline BRCA1/2-mutated (BRCA-MUT) HER2-negative locally advanced/metastatic breast cancer patients. BRCA-MUT tumors constitute 15-20% of triple-negative breast cancers (TNBCs). TNBCs are an aggressive subtype and lack overexpression of hormone receptors and HER2. Here, we evaluate the impact of the combination of talazoparib and a chemotherapeutic agent, carboplatin, to better understand which TNBCs will benefit from this combination, and the manner in which different sequencing strategies can impact primary tumor growth and distant metastasis development. Methods: We performed a 10-day chemosensitivity assay using 7 BRCA-MUT and BRCA wild-type TNBC cell lines. We tested 9 concentrations of talazoparib (Pfizer), and carboplatin (Selleckchem) each. Post-treatment, we used automated high-content imaging with Operetta (Perkin Elmer). We calculated IC50 values for talazoparib alone, CI (Combination Index) values, the DNA damage response (product of the percentage of cells positive for 53BP1 and mean 53BP1 foci per cell), and the apoptotic index (percentage of cells positive for cleaved-PARP). We also used an orthotopic xenograft model of MDAMB231 in NSG (NOD scid gamma) mice, with 8-14 mice in each treatment group. We treated mice with carboplatin (C) (35 mg/kg intraperitoneally) in combination with talazoparib (T) (0.03 mg/kg oral gavage) using 2 dosing strategies: a) concomitant administration of C + T; and b) T first, followed by C three days later, each compared to vehicle control. Results: We found a synergistic effect of the combination of talazoparib and carboplatin (CI &amp;lt; 1) in all 7 cell lines, with the greatest synergy (CI&amp;lt; 0.65) identified in three cell lines that were PARPi (PARP inhibitor)-resistant, namely HCC1143, MDAMB231, and Hs578T. For these three cell lines, we found that the mean 53BP1 product score increased by 7-16 fold in combination versus talazoparib alone at 0.2 µM, and the apoptotic index also increased by 4-26 fold at the same conditions. Concomitant administration of talazoparib and carboplatin in the MDAMB231 xenograft resulted in a 53.1% inhibition in primary tumor volume in comparison to control (P=0.0004), whereas sequential administration resulted in a 69.2% primary tumor volume inhibition (P&amp;lt;0.0001). The talazoparib first combination approach also resulted in a 53.9% decrease in lung micrometastasis (P=0.0003). Conclusion: The combination of talazoparib and carboplatin demonstrated a synergistic effect in 7 TNBC cell lines. We found greater DNA damage and cell death amongst PARPi-resistant cell lines and at lower concentrations of talazoparib when combined with carboplatin. In-vivo, sequential administration of talazoparib and carboplatin was the most effective approach to inhibit primary tumor growth. Citation Format: Alexia Cotte, Michèle Beniey, Takrima Haque, Nelly Béchir, Audrey Hubert, Korotoum W. Diallo, Danh Tran-Thanh, Saima N. Hassan. Pre-clinical combination of a PARP inhibitor, talazoparib, and carboplatin in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1066.

Abstract PS17-55: Reactive oxygen species scavengers in triple negative breast cancer

Abstract Reactive oxygen species (ROS) are well known to play important roles in cancer. In particular, ROS of mitochondrial origin (mtROS) promote DNA mutations and cell death when produced at high levels, but rather stimulate autophagy and metastatic spread when produced at subcytotoxic levels [1]. Multiple metabolic pathways and oncogenic regulators linked to ROS have been studied in cancer cells, including MAPK/ERK, PI3K/AKT, PTEN, PKD, BREF2, HIF-1, NF-κB and p53 [2]. Abnormalities in these pathways can contribute to tumor development.By producing mtROS, mitochondria control and could even trigger metastatic spread, meaning that the use of specific mitochondria-targeted superoxide inhibitors/scavengers, such as mitoquinol mesylate (MitoQ), could reduce and/or prevent metastatic dissemination. This has been studied and reported with the use of MitoTempo, which prevents metastasis of naturally metastatic MDA-MB-231 human breast cancer cells implanted in mice [1]. Here, we show that MitoQ impairs MDA-MB-231 and SkBr3 human breast cancer cell migration and invasion in vitro, and MDA-MB-231 metastasis to the lungs in mice in vivo. In metastatic take assays, tail vein-injected MDA-MB-231 cells pretreated with MitoQ formed significantly less metastases after 4 weeks compared to vehicle. In spontaneous metastatic assays, mice bearing orthotopic MDA-MB-231 tumors were treated with ± 20 mg/kg MitoQ (per os) for 6 weeks. Primary tumors were surgically removed to allow metastatic outgrowth. In this model, MitoQ decreased primary tumor recurrence as well as the number of lung micro-metastases. Additionally, RNAseq studies in two human breast cancer cell lines identified that MitoQ significantly modulated a common set of metabolic genes in vitro and in vivo. They can be used as biomarkers of response. These results, together with a successful Phase I clinical trial and the fact that MitoQ does not interfere with the cytotoxic effects of chemotherapy, confirm that MitoQ is a potential candidate to be clinically tested in triple negative breast cancer patients.

Abstract 4917: Estrogen receptor D538G mutation promotes cell migration via hyperactivation of Wnt signaling pathway

Abstract Background: Hotspot estrogen receptor-α (ERα/ESR1) mutations occur in 30-40% endocrine resistant ER+ breast cancer, with Y537S and D538G as the two most frequent hotspot mutations. A mostly unanswered question is and how these mutations facilitate metastatic processes. Methods: Frequencies of different ESR1 hotspot mutations were compared within three publicly available cohorts. Y537S and D538G genome-edited MCF7 and T47D cell models were used for in vitro characterization. Wound scratching, spheroid collective migration, chemotaxis assays were applied to examine different metastatic properties. Tail vein injection in nude mice followed by human CK19 lung section immune staining was used to characterize in vivo metastasis. Canonical Wnt pathway activity was studied using top-flash reporter assays, immunoblotting, and overexpression of dominant negative TCF4. ChIP-seq and ATAC-seq were utilized to profile ER global binding patterns and chromatin accessibility in cell models, respectively. Porcupine inhibitor-LGK974 was used to evaluate the efficiency of Wnt-targeted therapy. Results: D538G mutations were more frequent in ER+ metastatic lesions compared to Y537S among three metastatic breast cancer cohorts with unbiased SNV detection. In line with this, T47D-D538G ESR1 mutant cells showed strongly enhanced cell migration in vitro which was not observed in Y537S cells. We also observed increased lung micro-metastasis in vivo after tail vein injection of the D538G cells. Cell line transcriptomic analysis revealed uniquely hyperactivated Wnt pathway in D538G mutant cells, which was further confirmed in vitro by top-flash reporter and immunoblot. Suppression of canonical Wnt pathways blocked T47D-D538G specific cell migration. Combination treatment of fulvestrant and LGK974 synergistically inhibited D538G specific migration. Mechanistically, multiple Wnt regulator genes were found uniquely upregulated in D538G cells. Interestingly, none of these targets gained ER binding peaks at proximal regulatory regions, suggesting potential epigenetic regulation, which was confirmed in ATAC-seq results. Knockdown of FOXA1, a well-characterized pioneer factor, decreased canonical Wnt activity and abrogated the D538G-unique cell migration in short-term. Conclusion: T47D-D538G cells showed uniquely enhanced cell migration via Wnt hyperactivation, potentially mediated via epigenetic remodeling. These findings suggest the further study of potential combinatorial targeting of Wnt and ER signaling in D538G ESR1 mutant tumors. Citation Format: Zheqi Li, Kevin M. Levine, Spencer Arneson, Kristofer C. Berrett, Nolan M. Priedigkeit, Amir Bahreini, Jian Chen, Li Zhu, Jason S. Carroll, George C. Tseng, Peter C. Lucas, Jennifer M. Atkinson, Jason Gertz, Adrian V. Lee, Steffi Oesterreich. Estrogen receptor D538G mutation promotes cell migration via hyperactivation of Wnt signaling pathway [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 4917.

Chemogenomic profiling of breast cancer patient-derived xenografts reveals targetable vulnerabilities for difficult-to-treat tumors

Subsets of breast tumors present major clinical challenges, including triple-negative, metastatic/recurrent disease and rare histologies. Here, we developed 37 patient-derived xenografts (PDX) from these difficult-to-treat cancers to interrogate their molecular composition and functional biology. Whole-genome and transcriptome sequencing and reverse-phase protein arrays revealed that PDXs conserve the molecular landscape of their corresponding patient tumors. Metastatic potential varied between PDXs, where low-penetrance lung micrometastases were most common, though a subset of models displayed high rates of dissemination in organotropic or diffuse patterns consistent with what was observed clinically. Chemosensitivity profiling was performed in vivo with standard-of-care agents, where multi-drug chemoresistance was retained upon xenotransplantation. Consolidating chemogenomic data identified actionable features in the majority of PDXs, and marked regressions were observed in a subset that was evaluated in vivo. Together, this clinically-annotated PDX library with comprehensive molecular and phenotypic profiling serves as a resource for preclinical studies on difficult-to-treat breast tumors.