Metastatic Breast Cancer Model Research Articles

Structure-Activity Relationship Study Identifies a Novel Lipophilic Amiloride Derivative that Efficiently Kills Chemoresistant Breast Cancer Cells.

Derivatives of the potassium-sparing diuretic amiloride are preferentially cytotoxic toward tumor cells relative to normal cells, and have the capacity to target tumor cell populations resistant to currently employed therapeutic agents. However, a major barrier to clinical translation of the amilorides is their modest cytotoxic potency, with estimated IC 50 values in the high micromolar range. Here we report the synthesis of ten novel amiloride derivatives and the characterization of their cytotoxic potency toward MCF7 (ER/PR-positive), SKBR3 (HER2-positive) and MDA-MB-231 (triple negative) cell line models of breast cancer. Comparisons of derivative structure with cytotoxic potency toward these cell lines underscore the importance of an intact guanidine group, and uncover a strong link between drug-induced cytotoxicity and drug lipophilicity. We demonstrate that our most potent derivative called LLC1 is preferentially cytotoxic toward mouse mammary tumor over normal epithelial organoids, acts in the single digit micromolar range on breast cancer cell line models representing all major subtypes, acts on cell lines that exhibit both transient and sustained resistance to chemotherapeutic agents, but exhibits limited anti-tumor effects in a mouse model of metastatic breast cancer. Nonetheless, our observations offer a roadmap for the future optimization of amiloride-based compounds with preferential cytotoxicity toward breast tumor cells.

ONECUT2 is a druggable driver of luminal to basal breast cancer plasticity.

Tumor heterogeneity complicates patient treatment and can be due to transitioning of cancer cells across phenotypic cell states. This process is associated with the acquisition of independence from an oncogenic driver, such as the estrogen receptor (ER) in breast cancer (BC), resulting in tumor progression, therapeutic failure and metastatic spread. The transcription factor ONECUT2 (OC2) has been shown to be a master regulator protein of metastatic castration-resistant prostate cancer (mCRPC) tumors that promotes lineage plasticity to a drug-resistant neuroendocrine (NEPC) phenotype. Here, we investigate the role of OC2 in the dynamic conversion between different molecular subtypes in BC. We analyze OC2 expression and clinical significance in BC using public databases and immunohistochemical staining. In vitro, we perform RNA-Seq, RT-qPCR and western-blot after OC2 enforced expression. We also assess cellular effects of OC2 silencing and inhibition with a drug-like small molecule in vitro and in vivo. OC2 is highly expressed in a substantial subset of hormone receptor negative human BC tumors and tamoxifen-resistant models, and is associated with poor clinical outcome, lymph node metastasis and heightened clinical stage. OC2 inhibits ER expression and activity, suppresses a gene expression program associated with luminal differentiation and activates a basal-like state at the gene expression level. We also show that OC2 is required for cell growth and survival in metastatic BC models and that it can be targeted with a small molecule inhibitor providing a novel therapeutic strategy for patients with OC2 active tumors. The transcription factor OC2 is a driver of BC heterogeneity and a potential drug target in distinct cell states within the breast tumors.

Digital droplet PCR analysis of organoids generated from mouse mammary tumors demonstrates proof-of-concept capture of tumor heterogeneity.

Breast cancer metastases exhibit many different genetic alterations, including copy number amplifications (CNA). CNA are genetic alterations that are increasingly becoming relevant to breast oncology clinical practice. Here we identify CNA in metastatic breast tumor samples using publicly available datasets and characterize their expression and function using a metastatic mouse model of breast cancer. Our findings demonstrate that our organoid generation can be implemented to study clinically relevant features that reflect the genetic heterogeneity of individual tumors.

Isolation and Characterization of a Novel Mammary Adenocarcinoma, MCa-P1362, with Hormone Receptor Expression, Human Epidermal Growth Factor Receptor 2 Positivity, and Enrichment in Cancer and Mesenchymal Stem Cells

Preclinical models that display spontaneous metastasis are necessary to improve the therapeutic options for hormone receptor-positive breast cancers. Within this study, detailed cellular and molecular characterization was conducted on MCa-P1362, a newly established mouse model of metastatic breast cancer that is syngeneic in BALB/c mice. MCa-P1362 cancer cells express estrogen receptor, progesterone receptor, and the human epidermal growth factor receptor 2. MCa-P1362 cancer cells proliferate invitro and invivo in response to estrogen, yet do not depend on steroid hormones for growth and tumor progression. Analysis of MCa-P1362 tumor explants revealed the tumors contained a mixture of cancer cells and mesenchymal stromal cells. Through transcriptomic and functional analyses of both cancer and stromal cells, stem cells were detected within both populations. Functional studies demonstrated that MCa-P1362 cancer stem cells drove tumor initiation, whereas stromal cells from these tumors contributed to drug resistance. MCa-P1362 may serve as a useful preclinical model to investigate the cellular and molecular basis of breast tumor progression and therapeutic resistance.

Empagliflozin drives ferroptosis in anoikis-resistant cells by activating miR-128-3p dependent pathway and inhibiting CD98hc in breast cancer

A tumour suppressor miRNA, miR-128-3p, is widely involved in various biological processes and has been found to get downregulated in breast cancer patients. We previously published that ectopically expressed miR-128-3p suppressed migration, invasion, cell cycle arrest, and breast cancer stem cells. In the present study, we explored the role of Empagliflozin (EMPA) as a miR-128-3p functionality-mimicking drug in inducing ferroptosis by inhibiting CD98hc. Given that CD98hc is one of the proteins critical in triggering ferroptosis, we confirmed that miR-128-3p and EMPA inhibited SP1, leading to inhibition of CD98hc expression. Further, transfection with siCD98hc, miR-128-3p mimics, and inhibitors was performed to assess their involvement in the ferroptosis of anoikis-resistant cells. We proved that anoikis-resistant cells possess high ROS and iron levels. Further, miR-128-3p and EMPA treatments induced ferroptosis by inhibiting GSH and enzymatic activity of GPX4 and also induced lipid peroxidation. Moreover, EMPA suppressed bioluminescence of 4T1-Red-FLuc induced thoracic cavity, peritoneal tumour burden and lung nodules in an in-vivo metastatic model of breast cancer. Collectively, we revealed that EMPA sensitized the ECM detached cells to ferroptosis by synergically activating miR-128-3p and lowering the levels of SP1 and CD98hc, making it a potential adjunct drug for breast cancer chemotherapy.

Abstract LB174: OKI-219 is an inhibitor of PI3Kα H1047R that has brain penetrance and anti-tumor activity in a preclinical intracranial model of metastatic breast cancer

Abstract Background: Dysregulation of PI3K signaling is a common driver of solid tumors, with the PI3KαH1047R activating mutation found in 4% of all cancers. Because many cancers, including breast and lung cancers, can metastasize to the CNS, brain penetration should be a key feature of new therapies. The FDA-approved PI3K inhibitor alpelisib lacks CNS activity, and brain-penetrant alternatives such as buparlisib and paxalasib were not approved due to toxicity associated with a lack of mutant selectivity. OKI-219 is a potent, highly selective PI3KαH1047R inhibitor that has shown preclinical anti-tumor activity in multiple cancer models, both as a monotherapy and in combination with HER2- and ER-mediated therapeutics. Here, we demonstrate its brain penetrance across multiple animal species and anti-tumor activity in an intracranial mouse model of PI3KαH1047R mutant breast cancer. Materials and methods: Brain penetrance in mouse, rat, dog, and monkey was quantified by the ratio of unbound drug concentrations in brain to plasma (Kp,uu). Plasma, brain, and cerebrospinal fluid (CSF) concentrations of OKI-219 were measured by LC/MS, and protein binding in plasma, CSF, and brain homogenate were measured by rapid equilibrium dialysis. Additionally, free drug levels in the brain were directly measured by microdialysis in rats. Mice were implanted subcutaneously and intracranially with PI3KαH1047R xxT47D-luc tumors and tumor growth was measured by calipers (subcutaneous tumors) and luminescence (intracranial tumors). Results: At the Cmax of a pharmacologically active 10 mg/kg PO dose, OKI-219 had a Kp,uu of 0.34, 0.61, and 0.58 in rat, dog, and monkey CSF, respectively. Additionally, OKI-219 had an average Kp,uu of 0.67 over 24h post-dose as measured by brain microdialysis in rats at 30 mg/kg, with a dose-proportionate increase in brain free drug levels at 300 mg/kg. After 21 days of treatment, OKI-219 induced a statistically significant dose-dependent anti-tumor effect on both flank and brain xxT47D-luc tumors. Conclusion: Here we show that OKI-219 had consistently good brain penetrance across multiple species by multiple methods of measuring free drug levels in the brain. We further demonstrated that OKI-219 had anti-tumor activity against xxT47D-luc tumors in an intracranial mouse model that is similar to activity in the same tumor when implanted subcutaneously, demonstrating that the brain levels achieved were pharmacologically active. These data indicate the potential for activity of OKI-219 in CNS disease settings, including metastatic brain cancer, which are not currently addressed with existing PI3K pathway inhibitors. OnKure has initiated the PIKture Phase I clinical study (OKI-219-101) to evaluate OKI-219 in patients with PI3KαH1047R-mutated solid tumors. Citation Format: Alexander A. Strait, Molly A. Taylor, Kevin S. Litwiler, Qian Zhao, David A. Mareska, Mark L. Boys, Yevgeniy Izrayelit, Richard Woessner, James D. Winkler. OKI-219 is an inhibitor of PI3Kα H1047R that has brain penetrance and anti-tumor activity in a preclinical intracranial model of metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB174.

Abstract LB355: Cryo-immune vaccination (CIV) by SYNC-T therapy: Preclinical modeling of a novel device-multidrug immunotherapeutic approach to eradicate advanced metastatic cancers

Abstract A major challenge to further development of immune checkpoint therapies (ICT) for advanced cancer is the inherent toxicity of higher-order multidrug combinations likely needed to eradicate metastatic disease. Recently, early clinical studies have investigated a new general approach to address this challenge, termed cryo-immune vaccination (CIV). The CIV approach by SYNC-T therapy involves cycles of local cryolysis of a solid tumor followed by infusion of the same lesion with a low-dose multi-API formulation. CIV has never been explored methodically in preclinical or clinical settings, so the basis for systemic responses that have been observed in early clinical testing are not fully understood. Accordingly, as trials of the safety and efficacy of CIV proceed, there is now a need of preclinical model systems to help illuminate the mechanistic foundations of the approach, investigate safety and efficacy, and learn where and how it might be best leveraged. To develop the conceptual framework of CIV, we adapted the orthotopic metastatic 4T1 breast cancer model used extensively in our laboratory as a foundation to study CIV antitumor responses and mechanisms of action. Briefly, breast tumors were initiated in naïve murine subjects by orthotopic injection of 1x104 cells into the mammary fad pads of immunocompetent BALB/c hosts. When tumors reached ~5-7 mm, a time when occult pulmonary metastases have seeded, primary tumors were exposed surgically on skin flaps and contacted directly with a liquid nitrogen-cooled pointed steel rod for 15-30 sec. The rod was then withdrawn and tumor tissue allowed to thaw fully before injection into the cryolysed tumor of a selected ICT multidrug cocktail (or negative control murine IgG) in 25-50 µl total volume. The selected multidrug cocktail was modeled on SYNC-T combination therapy currently under study in a Phase 1 clinical trial (Syncromune, Inc.) Briefly, the ICT drug cocktails were variably composed of murine-reactive antibodies against CTLA4, PD-1 or OX40 (agonist), CD40L (agonist) and CpG oligonucleotide. As a starting point, allometric scaling was used to model dosing analogous to the proprietary ICT drug formulations SV-101 and SV-102 in clinical testing (Syncromune, Inc.). For exploratory toxicology, a standard panel of blood-borne liver and kidney toxicity biomarkers was assessed. For exploratory pharmacology, a ‘super mouse’ design was employed with ELISA-based determinations to measure antibody distribution in blood serum. To gain initial mechanistic insights, responses will be compared in host mice that are genetically deficient in selected components of the adaptive or innate immune systems. Progress in methodological and mechanistic studies will be presented. Citation Format: James B. DuHadaway, Alexander J. Muller, Lisa D. Laury-Kleintop, U. Margaretha Wallon, Susan K. Gilmour, Marie Webster, Jason R. Williams, Gabriela R. Rossi, Mario R. Mautino, Jonathan Lewis, Charles J. Link, George C. Prendergast. Cryo-immune vaccination (CIV) by SYNC-T therapy: Preclinical modeling of a novel device-multidrug immunotherapeutic approach to eradicate advanced metastatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB355.

Iron Oxide Nanoparticles Inhibit Tumor Progression and Suppress Lung Metastases in Mouse Models of Breast Cancer.

Systemic exposure to starch-coated iron oxide nanoparticles (IONPs) can stimulate antitumor T cell responses, even when little IONP is retained within the tumor. Here, we demonstrate in mouse models of metastatic breast cancer that IONPs can alter the host immune landscape, leading to systemic immune-mediated disease suppression. We report that a single intravenous injection of IONPs can inhibit primary tumor growth, suppress metastases, and extend survival. Gene expression analysis revealed the activation of Toll-like receptor (TLR) pathways involving signaling via Toll/Interleukin-1 receptor domain-containing adaptor-inducing IFN-β (TRIF), a TLR pathway adaptor protein. Requisite participation of TRIF in suppressing tumor progression was demonstrated with histopathologic evidence of upregulated IFN-regulatory factor 3 (IRF3), a downstream protein, and confirmed in a TRIF knockout syngeneic mouse model of metastatic breast cancer. Neither starch-coated polystyrene nanoparticles lacking iron, nor iron-containing dextran-coated parenteral iron replacement agent, induced significant antitumor effects, suggesting a dependence on the type of IONP formulation. Analysis of multiple independent clinical databases supports a hypothesis that upregulation of TLR3 and IRF3 correlates with increased overall survival among breast cancer patients. Taken together, these data support a compelling rationale to re-examine IONP formulations as harboring anticancer immune (nano)adjuvant properties to generate a therapeutic benefit without requiring uptake by cancer cells.

Abstract 3326: PMR-116, a second generation RNA Polymerase I inhibitor

Abstract Introduction: Inhibition of RNA Polymerase I, the enzyme responsible for the synthesis of ribosomal RNAs, the nucleic scaffold of ribosomes, has been proven as a novel therapeutic target as most cancer cells are "addicted" to ribosome biogenesis. Methods: In collaboration with Pimera Inc, we developed and tested the efficacy of a second generation RNA polymerase I inhibitor, PMR-116, in various pre-clinical cancer models. On-target activity of PMR-116 was confirmed in peripheral blood cells isolated from patients with solid tumors enrolled in a phase I trial of PMR-116. Results: PMR-116 potently inhibits Pol I transcription with ~200 fold higher selectivity towards Pol I compared to Pol II transcription. PMR-116 inhibits rRNA synthesis by stalling the Pol I complex at the rDNA promoter preventing promoter escape and consequently elongation. PMR-116 anti-cancer activity was evaluated against a panel of over 100 cancer cell lines representative of 20 major types of solid and haematological malignancies. This new Pol I inhibitor exhibited broad anti-proliferative and cytotoxic activity with an IC50 ranging from 32-4500nM and a median IC50 of 300nM. In contrast, cells derived from normal tissues were significantly less sensitive (IC50 ranging from 6-33μM). In vivo, PMR-116 significantly improved survival and reduce tumor burden in several preclinical models such as Vk*MYC transgenic model of indolent multiple myeloma, CT26 xenograft model of colorectal cancer, MMTV-PyMT transgenic model of metastatic breast cancer, mixed-lineage leukemia (MLL)-eleven nineteen leukemia (ENL)+Nras acute myeloid leukemia (+/-p53 mutation) and patient-derived xenografts of prostate cancer and AML. In a phase I trial of PMR-116, on-target activity of PMR-116 was observed in cells derived from the peripheral blood. Conclusion: PMR-116 significantly reduced cancer growth in a wide range of tumor models, including GEMM, syngeneic and PDX models. PMR-116 is undergoing clinical testing in phase I in solid tumors with the objective to determine maximum tolerated dose and identify a phase II dose. Citation Format: Luc Furic, Nadine Hein, Rita Ferreira, Konstantin Panov, Alisee Huglo, Richard Rebello, Eric Kusnadi, Denis Drygin, Mustapha Haddach, Ross Hannan. PMR-116, a second generation RNA Polymerase I inhibitor [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 3326.

Abstract 5451: FOXC2 influences EMT-induced stem cell properties through regulation of FABP4 in breast cancer

Abstract Cancer stem cells (CSCs) pose a significant clinical challenge due to their inherent resistance to therapies and prevalence in cancer recurrence. CSCs have the ability for self-renewal and to adapt to different microenvironments during dissemination and seeding into distal organs, and these properties are critical in metastasis. We and others have shown that epithelial to mesenchymal transition (EMT) drives the acquisition of stem cell properties, particularly via the forkhead box protein C2 (FOXC2) transcription factor. FOXC2 is a master regulator of EMT and is necessary for the induction and maintenance of CSC properties. Importantly, FOXC2 is not expressed in most adult tissues except adipocytes, in which it dictates several aspects of lipid metabolism. Additionally, metabolic changes during EMT can equip cancer cells with enhanced survival mechanisms during tumor progression and metastasis. We hypothesized that FOXC2 regulates metabolism in EMT-induced cells, facilitating stem cell properties and metastatic competence. To determine the downstream metabolic genes regulated by FOXC2 and that enhance stemness properties in cancer cells, we performed gene expression profiling and identified fatty acid binding protein 4 (FABP4), which is known to be involved in lipid metabolism, especially in adipocytes and macrophages. We demonstrate that inhibition of FOXC2 expression using shRNA in EMT-induced cells markedly reduces FABP4 expression. In agreement with this, we found that the overexpression of FOXC2 expression rescues the expression of FABP4. In addition, FOXC2 binds to the FABP4 promoter and regulates its expression suggesting that FABP4 is a direct target of FOXC2. We demonstrate that both lipid uptake and CSC formation is inhibited in a dose-dependent manner using a FABP4-specific small molecule inhibitor. Additional experiments are underway to assess the role of FABP4 in CSC formation in vivo and in contributions to metastasis, using our small molecule inhibitor in mouse models of metastatic breast cancer. Overall, our data indicate that FABP4 is a key regulator of stemness in breast cancer cells and targeting the FOXC2—FABP4 axis may provide therapeutic benefits for breast cancer. This work also provides insights into the metabolic shifts that occur during EMT and CSC formation, which warrants further investigation into these mechanisms. Citation Format: Breanna R. Demestichas, Vinita Shivakumar, Esmeralda Ramirez-Peña, Joseph Taube, Petra den Hollander, Sendurai A. Mani. FOXC2 influences EMT-induced stem cell properties through regulation of FABP4 in breast cancer [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 5451.

Abstract 5895: N17350 kills cancer cells, spares immune cells, and regresses CDX tumors from chemotherapy naive and experienced patients

Abstract Background. While multiple studies have demonstrated improved clinical outcomes in patients receiving concurrent chemotherapy and immunotherapy, clinical synergy may be limited by the destructive effects of DNA-damaging chemotherapy on non-cancer cells, including key constituents of anti-tumor immunity (e.g. mature T cells). Furthermore, when first-line chemotherapy fails, subsequent lines of chemotherapy produce lower response rates. Thus, new approaches that eradicate cancer cells while preserving immune cells may be required to more efficiently induce anti-tumor immunity as monotherapy or in combination with immunotherapy. Neutrophil elastase (ELANE), a neutrophil-derived serine protease, kills a wide range of cancer cells without harming immune cells through a unique mechanism involving histone H1 and the death domain of CD95. Here, we compared N17350, an optimized biologic leveraging the ELANE pathway, and chemotherapies for their ability to kill cancer cells and preserve immune cells from primary tumors of ovarian cancer (OvCa) patients and evaluated efficacy in OvCa patient cell-derived xenografts (CDXs) and a 4T1 model of metastatic breast cancer. Methods. For in vitro studies, cancer cells and CD45+ immune cells were isolated from primary tumors of chemotherapy naïve and experienced OvCa patients. Cells were treated with N17350, oxaliplatin, or doxorubicin 24h post-isolation, and viability was quantified by calcein-AM. For in vivo studies, primary OvCa cells were injected into nude mice to create CDX models. Mice were treated with 2 doses of N17350 (400μg/100mm3, intratumoral) or carboplatin (100mg/kg, intraperitoneal), and effects on tumor growth were quantified. In 4T1 model, N17350 was compared to oxaliplatin, alone or in combination with anti-CTLA4, and effects on tumor growth and immune profile were assessed. Results. N17350 killed primary cancer cells from all OvCa patients tested but was well tolerated by CD45+ tumor immune cells from the same patients. In contrast, doxorubicin and oxaliplatin showed similar toxicity to both cell types. N17350 killed cancer cells from chemotherapy naïve and experienced patients with equal efficacy, while doxorubicin and oxaliplatin showed less efficacy in patients previously treated with chemotherapy. N17350 rapidly regressed tumors in all OvCa CDX models and exhibited markedly improved efficacy over carboplatin. In the 4T1 model, N17350 outperformed oxaliplatin in terms of regressing tumors, elevating tumor immune cells, and combining with anti-CTLA4. Conclusions. Our data demonstrate that N17350 kills OvCa tumors in vitro and in vivo while sparing CD45+ immune cells in both chemotherapy naïve and experienced patients. Together with previous data showing that N17350 induces immunogenic tumor cell death, these findings suggest that N17350 could improve clinical outcomes compared to chemoimmunotherapy. Citation Format: Chang Cui, Ravindra Gujar, Christine Lee, Maria Fumagalli, Nicole Martinez, Hannah Liu, Nicole Grigaitis, Sonia Feau, Afshin Bahador, Alain P. Algazi, Lev Becker. N17350 kills cancer cells, spares immune cells, and regresses CDX tumors from chemotherapy naive and experienced patients [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 5895.

Abstract 6521: Macrophage-targeting immunotherapy enhances chemotherapy response in primary and metastatic triple-negative breast cancer models

Abstract Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks targeted treatment options. While immune checkpoint blockade leads to significant benefits in the treatment of TNBC when in combination with chemotherapy, recurrence, metastasis, and chemoresistance still remain major challenges. The purpose of this study was to assess the ability of a colony-stimulating factor 1 receptor inhibitor (CSF-1Ri) to shift the phenotype of the tumor microenvironment (TME) in murine models of TNBC, and the potential benefit of such in enhancing standard of care chemotherapy. We focused on targeting tumor-associated macrophages (TAMs) as they are the most abundant immune infiltrate in the TNBC TME and are associated with worsening overall survival (OS) and metastasis free survival (MFS). Targeting TAMs, specifically M2-like TAMs, has been shown to inhibit tumor progression and improve OS in the clinic. We selected pexidartinib (PLX) as the CSF-1Ri of choice, given its translational potential (FDA approved) and gemcitabine (GEM) as chemotherapy, given its ability to target myeloid derived suppressor cells and M2-like TAMs. For primary TNBC we employed 4T1 mammary carcinoma cells transfected with luciferase reporter gene in BALB/c mice as an immunocompetent model. Mice were injected with 100,000 cells in the 4th mammary fat pad, and tumors established over the course of fifteen days. Mice were then exposed to daily administrations of PLX (8 mg/kg), and weekly administrations of GEM (60 mg/kg). Tumor volumes were recorded every other day. The TME was analyzed with flow cytometry (FC), immunohistochemistry (IHC), and multiplex immunofluorescence (mIF). For the metastatic model, mice were inoculated with 100,000 4T1-luc cells in the flank. On day six, mice were exposed to treatments as outlined above. Mice underwent survival surgery to remove the primary tumor on day thirteen and were then treated with adjuvant therapy. Metastasis and OS were tracked. FC analysis indicated only combination therapy (11.8% ± 2.1) significantly decreased total TAMs compared to vehicle (22% ± 2) in the primary tumor. Combination therapy also showed the greatest depletion of M2-like TAMs (2.2% ± 0.6), with a significant decrease compared to both vehicle (15.6% ± 0.4) and GEM (6.6% ± 0.9). This TME modulation was confirmed using IHC and mIF. After four cycles of treatment, combination therapy (232.9 mm3 ± 130.8) had a significant effect on tumor burden compared to GEM alone (369.7 mm3 ± 165.8), indicating a correlation between tumor phenotype and therapeutic benefit. Furthermore, combination therapy led to the greatest prolongation of MFS and OS in the resection model, indicating that combination therapy has efficacy in the neoadjuvant and adjuvant setting. In conclusion, targeting TAMs in TNBC primes the tumor for combination with chemotherapy in TNBC. Citation Format: Matthew Emilio Fernandez, Yasir Alshehry, Laura Graham, Jennifer E. Koblinski, Harry D. Bear, Doug H. Sweet, Sandro R. da Rocha. Macrophage-targeting immunotherapy enhances chemotherapy response in primary and metastatic triple-negative breast cancer models [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 6521.

Abstract 2770: A novel MIRO2/MYO9B/RhoA signaling axis controls tumor cell invasion and metastasis

Abstract Metastasis of cancer cells to vital organs remains the leading cause of cancer related deaths, emphasizing a strong need for actionable targets in advanced stage cancer. To address this, we study novel dysregulated mitochondrial signaling mechanisms that cells utilize to metastasize. Here, we focus on how outer mitochondrial membrane protein—Mitochondrial Rho GTPase 2 (MIRO2)—promotes tumor cell invasion and metastasis. Our previous work identified higher MIRO2 mRNA expression in cancer vs. normal patient samples in a multitude of cancer types, which correlated with worse patient outcomes. Furthermore, we demonstrated that MIRO2 was critical for prostate cancer cell growth and survival in vitro and in vivo. However, it remains unknown if MIRO2 only affects primary tumor growth or if this protein is important throughout tumor progression. Using siRNA mediated knockdown (KD) of MIRO2 we find MIRO2 KD ubiquitously reduces tumor cell invasion in breast, melanoma, pancreatic, and prostate cancer cells. Utilizing metastatic prostate and breast cancer models, we demonstrate that mice injected with MIRO2 shRNA cells have significantly lower metastatic burden compared to mice injected with control shRNA cells. To determine the mechanism that MIRO2 controls invasion and metastasis, we looked at which of the top protein-binding partners for MIRO2 may regulate tumor cell invasion. Of our top hits, we found that depletion of atypical myosin IXB (MYO9B) by siRNA most significantly reduced tumor cell invasion and phenocopied MIRO2 depletion. MYO9B has a well-defined role in controlling cell motility via inactivation of RhoA. Excitingly, we have found 1) MIRO2 KD results in increased active RhoA, phenocopying MYO9B KD, 2) dual ablation of MIRO2 and RhoA fully rescues tumor cell invasion, and 3) MIRO2 is required for MYO9B driven invasion. Taken together, we propose a novel signaling mechanism by which MIRO2 broadly promotes invasion and metastasis through MYO9B dependent inactivation of RhoA. Citation Format: Dillon P. Boulton, Connor J. Hughes, Valentina Vaira, Ajita Jindal, Masoom Raza, Stephen Connor Purdy, Shilpa Sant, Heide L. Ford, M. Cecilia Caino. A novel MIRO2/MYO9B/RhoA signaling axis controls tumor cell invasion and 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 2770.

Effects of Tumor Microenvironment on the Primo Vascular Pattern in the Mouse Model of Metastatic Breast Cancer.

: Tumor survival, promotion, and metastatic functions are regulated by the tumor microenvironment (TME). The primo vascular system (PVS), the third circulatory system in animals, is currently thought to be a highly effective pathway for the spread of cancer cells. : In the present study, we intend to determine the TME effects on the PVS pattern in breast cancer for the first time. : Heterotopic and orthotopic metastatic triple-negative breast cancer (TNBC) mice models were created. After 35 days, the skin was retracted, and a 2 cm skin incision was made up and down from the surface of the tumor tissue. In preparation for PVS staining, the dyes (trypan blue and alamarBlue) were injected throughout the tumor tissues. Under a stereomicroscope, PVS in heterotopic and orthotopic tumors was seen. : According to our data, there are no appreciable variations in PVS patterns and density between heterotopic and orthotopic animal models. Furthermore, alamarBlue is a good option for tumor PVS staining, as demonstrated by our research. : For the first time, our data gave significant new information about the PVS in TNBC. Creating new anti-cancer treatments may be made possible by a better understanding of the biological characteristics of the TME and PVS.

Abstract B045: Novel Small Molecule Inhibitors Targeting Proinflammatory Cytokines in Metastatic Breast Cancer

Abstract Breast cancer is currently the most commonly diagnosed and second leading cause of cancer-related deaths among women in the Unites States. For the past several years, research has focused on the role of inflammatory cytokines such as interleukin-6 (IL-6) and their role in cancer. Increasing evidence suggests that breast cancer patients with high levels of proinflammatory modulators have an overall worse prognosis. Research performed by our lab and others has also shown that oncostatin M (OSM) promotes breast cancer progression and metastasis by inducing circulating tumor cell (CTC) numbers, and metastasis to distant sites in vivo. While efforts have been made to develop FDA approved therapeutics for OSM, previous studies have all utilized monoclonal antibodies and none have survived clinical trials. For the first time, our research group has developed a stepwise approach towards designing and testing novel small molecule inhibitors (SMIs) against OSM. To determine viable candidates, in silico computational screening of compound libraries towards OSM revealed top potential compounds that interact with the receptor binding region and inhibit biological function. These top candidate inhibitors were then analyzed for direct interaction with cytokines using fluorescent quenching and chemical shift perturbation NMR (CSP-NMR). In vitro efficacy of each SMI was then analyzed by measuring inhibition of all signaling cascades activated by OSM using enzyme-linked immunosorbent assays (ELISA) and immunoblot assays. From these in silico and in vitro methods, SMIs were then structurally modified with effective functional groups to increase efficacy of binding interaction and reduce toxicity for living systems. Finally, our most promising small molecule inhibitor that has been sufficiently tested for efficacy was examined in vivo utilizing a novel estrogen receptor-positive (ER+) mouse model of metastatic breast cancer, and treatment with our SMI proved to decrease tumor burden and metastasis, particularly to the lung when compared to vehicle control. In conclusion, our lab has generated the first ever SMI aimed to inhibit OSM-mediated cancer progression. Our continued efforts aim to develop a novel FDA approved therapeutic to improve outcomes for breast cancer patients. Citation Format: Cody L Wolf, Clyde Pruett, Darren Lighter, Cooper McGrath, Andrea Feci, Joseph Tuccinardi, Simion Dinca, Ken Tawara, Matthew King, Lisa Warner, Don Warner, Cheryl L. Jorcyk. Novel Small Molecule Inhibitors Targeting Proinflammatory Cytokines in Metastatic Breast Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B045.

Extracellular Matrix-Trapped Bioinspired Lipoprotein Prolongs Tumor Retention to Potentiate Antitumor Immunity.

The immunomodulatory effects of many therapeutic agents are significantly challenged by their insufficient delivery efficiency and short retention time in tumors. Regarding the distinctively upregulated fibronectin (FN1) and tenascin C (TNC) in tumor stroma, herein a protease-activated FN1 and/or TNC binding peptide (FTF) is designed and an extracellular matrix (ECM)-trapped bioinspired lipoprotein (BL) (FTF-BL-CP) is proposed that can be preferentially captured by the TNC and/or FN1 for tumor retention, and then be responsively dissociated from the matrix to potentiate the antitumor immunity. The FTF-BL-CP treatment produces a 6.96-, 9.24-, 6.72-, 7.32-, and 6.73-fold increase of CD3+CD8+ T cells and their interferon-γ-, granzyme B-, perforin-, and Ki67-expressing subtypes versus the negative control, thereby profoundly eliciting the antitumor immunity. In orthotopic and lung metastatic breast cancer models, FTF-BL-CP produces notable therapeutic benefits of retarding tumor growth, extending survivals, and inhibiting lung metastasis. Therefore, this ECM-trapping strategy provides an encouraging possibility of prolonging tumor retention to potentiate the antitumor immunity for anticancer immunotherapy.

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

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

A Novel Metastatic Estrogen Receptor-Expressing Breast Cancer Model with Antiestrogen Responsiveness.

Most women diagnosed with breast cancer (BC) have estrogen receptor alpha-positive (ER+) disease. The current mouse models of ER+ BC often rely on exogenous estrogen to encourage metastasis, which modifies the immune system and the function of some tissues like bone. Other studies use genetically modified or immunocompromised mouse strains, which do not accurately replicate the clinical disease. To create a model of antiestrogen responsive BC with spontaneous metastasis, we developed a mouse model of 4T1.2 triple-negative (TN) breast cancer with virally transduced ER expression that metastasizes spontaneously without exogenous estrogen stimulation and is responsive to antiestrogen drugs. Our mouse model exhibited upregulated ER-responsive genes and multi-organ metastasis without exogenous estrogen administration. Additionally, we developed a second TN BC cell line, E0771/bone, to express ER, and while it expressed ER-responsive genes, it lacked spontaneous metastasis to clinically important tissues. Following antiestrogen treatment (tamoxifen, ICI 182,780, or vehicle control), 4T1.2- and E0771/bone-derived tumor volumes and weights were significantly decreased, exemplifying antiestrogen responsivity in both cell lines. This 4T1.2 tumor model, which expresses the estrogen receptor, metastasizes spontaneously, and responds to antiestrogen treatment, will allow for further investigation into the biology and potential treatment of metastasis.

Single-cell Analysis Reveals Inter- and Intratumour Heterogeneity in Metastatic Breast Cancer

Metastasis is the leading cause of cancer-related deaths of breast cancer patients. Some cancer cells in a tumour go through successive steps, referred to as the metastatic cascade, and give rise to metastases at a distant site. We know that the plasticity and heterogeneity of cancer cells play critical roles in metastasis but the precise underlying molecular mechanisms remain elusive. Here we aimed to identify molecular mechanisms of metastasis during colonization, one of the most important yet poorly understood steps of the cascade. We performed single-cell RNA-Seq (scRNA-Seq) on tumours and matched lung macrometastases of patient-derived xenografts of breast cancer. After correcting for confounding factors such as the cell cycle and the percentage of detected genes (PDG), we identified cells in three states in both tumours and metastases. Gene-set enrichment analysis revealed biological processes specific to proliferation and invasion in two states. Our findings suggest that these states are a balance between epithelial-to-mesenchymal (EMT) and mesenchymal-to-epithelial transitions (MET) traits that results in so-called partial EMT phenotypes. Analysis of the top differentially expressed genes (DEGs) between these cell states revealed a common set of partial EMT transcription factors (TFs) controlling gene expression, including ZNF750, OVOL2, TP63, TFAP2C and HEY2. Our data suggest that the TFs related to EMT delineate different cell states in tumours and metastases. The results highlight the marked interpatient heterogeneity of breast cancer but identify common features of single cells from five models of metastatic breast cancer.

Formation of an invasion-permissive matrix requires TGFβ/SNAIL1-regulated alternative splicing of fibronectin

BackgroundAs in most solid cancers, the emergence of cells with oncogenic mutations in the mammary epithelium alters the tissue homeostasis. Some soluble factors, such as TGFβ, potently modify the behavior of healthy stromal cells. A subpopulation of cancer-associated fibroblasts expressing a TGFβ target, the SNAIL1 transcription factor, display myofibroblastic abilities that rearrange the stromal architecture. Breast tumors with the presence of SNAIL1 in the stromal compartment, and with aligned extracellular fiber, are associated with poor survival prognoses.MethodsWe used deep RNA sequencing and biochemical techniques to study alternative splicing and human tumor databases to test for associations (correlation t-test) between SNAIL1 and fibronectin isoforms. Three-dimensional extracellular matrices generated from fibroblasts were used to study the mechanical properties and actions of the extracellular matrices on tumor cell and fibroblast behaviors. A metastatic mouse model of breast cancer was used to test the action of fibronectin isoforms on lung metastasis.ResultsIn silico studies showed that SNAIL1 correlates with the expression of the extra domain A (EDA)-containing (EDA+) fibronectin in advanced human breast cancer and other types of epithelial cancers. In TGFβ-activated fibroblasts, alternative splicing of fibronectin as well as of 500 other genes was modified by eliminating SNAIL1. Biochemical analyses demonstrated that SNAIL1 favors the inclusion of the EDA exon by modulating the activity of the SRSF1 splicing factor. Similar to Snai1 knockout fibroblasts, EDA- fibronectin fibroblasts produce an extracellular matrix that does not sustain TGFβ-induced fiber organization, rigidity, fibroblast activation, or tumor cell invasion. The presence of EDA+ fibronectin changes the action of metalloproteinases on fibronectin fibers. Critically, in an mouse orthotopic breast cancer model, the absence of the fibronectin EDA domain completely prevents lung metastasis.ConclusionsOur results support the requirement of EDA+ fibronectin in the generation of a metastasis permissive stromal architecture in breast cancers and its molecular control by SNAIL1. From a pharmacological point of view, specifically blocking EDA+ fibronectin deposition could be included in studies to reduce the formation of a pro-metastatic environment.