Breast Cancer Model Research Articles

Abstract C037: Chemotherapy causes de novo induction of invasive breast cancer cells

Abstract Metastasis is the leading cause of breast cancer (BC) death, and tumor cells must migrate and invade to metastasize. BC cells that express the actin regulatory protein MenaINV have an enhanced ability to migrate and intravasate within the primary tumor, and extravasate at secondary sites. Though chemotherapy improves patient survival, treatment with paclitaxel, a chemotherapy used for BC, leads to upregulation of MenaINV and an increase in metastasis in mice. MenaINV expression can be induced in BC cells through Notch1 signaling with macrophages, which are often recruited to tumors in response to chemotherapy. MenaINV-expressing cells are also resistant to paclitaxel, begging the question of whether paclitaxel increases MenaINV by de novo induction or by selectively killing non-MenaINV-expressing cells. We hypothesized that paclitaxel causes de novo MenaINV induction by increasing macrophage-tumor cell Notch signaling. Understanding this pro-metastatic effect of chemotherapy is crucial to refining treatment strategies. MMTV-PyMT mice bearing spontaneous mammary tumors were used as a model of BC. Mice were treated with paclitaxel or vehicle every 5 days for a total of 3 treatments. To study the role of macrophages in the paclitaxel-mediated MenaINV increase, in addition to paclitaxel, one cohort of mice was co-administered the macrophage-depleting agent clodronate or vehicle. To study the role of Notch signaling, a second cohort of mice was co-administered the Notch inhibitor DAPT or vehicle. Immunofluorescence staining was used to evaluate tumors for: %MenaINV+ area, proportion of apoptotic cells, macrophage density, and macrophage expression of Notch ligands Jag1 and Jag2. Paclitaxel-treated tumors expressed significantly more MenaINV even when tumor cell death did not increase, indicating that chemotherapy increases MenaINV expression by induction. Both macrophages and Notch signaling were needed for this effect to occur. Interestingly, paclitaxel did not increase macrophage infiltration or macrophage expression of Notch ligands, suggesting that paclitaxel may increase MenaINV by acting on tumor cells to promote Notch signaling. Paclitaxel causes de novo MenaINV induction that is macrophage- and Notch-dependent. Studies aiming to further elucidate the mechanism behind paclitaxel-mediated MenaINV induction are ongoing. These results lay the groundwork for novel microenvironment-based therapies to alleviate the pro-metastatic effects of chemotherapy in BC. Citation Format: Madeline Friedman-DeLuca, George S. Karagiannis, Camille L. Duran, Luis Rivera Sanchez, Prachiben P. Patel, Suryansh Shukla, Nicole D. Barth, Ved P. Sharma, Michael Papanicolaou, John S. Condeelis, Maja H. Oktay, David Entenberg. Chemotherapy causes de novo induction of invasive breast cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C037.

Abstract A033: Integrating spatial transcriptomics and second harmonic generation imaging to identify targets of supermere-associated Discoidin Domain Receptor 1 involved in collagen alignment

Abstract Immune checkpoint blockade has been shown to be an effective treatment for colorectal cancers (CRCs) highly infiltrated by CD8+ T cells. However, there is a paucity of CD8+ T-cell infiltration in the majority of CRCs. We previously identified the collagen-activated receptor Discoidin Domain Receptor 1 (DDR1) as a member of a four-gene signature associated with T-cell exclusion in CRC. The cleaved ectodomain (cECD) of DDR1 induces collagen alignment, leading to T-cell exclusion in a breast cancer model. We found that almost all DDR1 cECD is found in supermeres, a novel secreted nanoparticle identified by our lab, and that DDR1 cECD is required for supermere signaling in recipient cells. We hypothesized that supermere-associated DDR1 cECD signals to CRC stromal cells, leading to collagen alignment. To test this hypothesis, we developed a method to determine which genes are associated with collagen alignment and DDR1 expression. To do this, 30 μm thick CRC sections are imaged using a Nikon AX multiphoton microscope by second harmonic generation imaging, which permits label-free imaging of collagen fibers. Spatial heatmaps of the collagen alignment are generated from the resulting images. Spatial transcriptomics is performed on a serial tumor section using the Visium v2 platform and the gene expression matrix is integrated with the collagen alignment heatmap data. As proof-of-principle, two CRC tumors have been analyzed using this method. Highly aligned regions were found to be associated with expression of genes involved in extracellular matrix organization, keloidal collagen and specific fibroblast subtypes. Genes which may be regulated by DDR1 cECD and be involved in collagen alignment will be confirmed at the protein level by multiplexed immunofluorescence on serial sections and in vitro experiments. This method will provide a rich dataset to elucidate the roles of supermeres and DDR1 cECD in T-cell exclusion and collagen fiber alignment and may support the discovery of new strategies to overcome immune exclusion in CRC. Citation Format: Maxwell S. Hamilton, Oleg Tutanov, Harsimran Kaur, Alan J. Simmons, Clark Massick, Kasey Vickers, Ken S. Lau, Ambra Pozzi, Robert J. Coffey. Integrating spatial transcriptomics and second harmonic generation imaging to identify targets of supermere-associated Discoidin Domain Receptor 1 involved in collagen alignment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A033.

Abstract C014: Degradation of extracellular trap DNA sustains anti-tumor immune responses in breast cancer

Abstract Increased extracellular DNA (exDNA) levels in the blood are a hallmark of metastatic cancer, arising from tumor lysis, apoptosis, necrosis, and neutrophil extracellular traps (NETs) released by tumor-expanded neutrophils. NETs, web-like chromatin structures extruded by neutrophils to trap pathogens, are highly immunogenic due to their DNA and histone content and their persistent interaction with dendritic cells. Tumor cells can also release extracellular traps. In myeloid leukemia, blasts release nuclear content via traps to activate coagulation or sustain myeloproliferation. Extracellular traps have been found in NPM1 mutant AML patients, with mutant NPM co-localizing with histones along exDNA traps. Forcing trap extrusion in NPM mutant leukemia cells has been used to create dendritic cell-based vaccines that break tolerance against NPMc antigens. This suggests that NETs in the tumor microenvironment (TME) may promote tolerance, with breaking tolerance requiring consistent NET formation and dendritic cell interaction. NETs are also seen in solid tumors, such as triple-negative breast cancer (TNBC), where they aid in cancer cell migration, invasion, and awakening of circulating tumor cells. However, the capacity of tumor cells to directly extrude DNA traps is not fully understood. To investigate this, various human and murine breast cancer cell lines were seeded on poly-D-lysine coated slides, stained with DAPI and Sytoxgreen, and observed via confocal microscopy. Tumor cells extruded DNA traps within 4 hours without stimuli. Notably, trap extrusion correlated with cell line aggressiveness and was abolished by DNase, indicating dsDNA composition. The absence of traps in 24-hour cultures suggests DNA extrusion may help rare tumor cells survive and proliferate. In line, DNase treatment reduced tumor proliferation and increased apoptosis. To assess the significance of DNA traps in vivo, BALB/c mice were injected with the highly metastatic 4T1 clone 5 breast cancer model, with or without DNase treatment, and monitored starting 5 days post-injection and weekly until day 28. Histological analysis at the injection site 5 days post-injection showed that DNase treatment reduced both local trap formation and tumor cell proliferation, as indicated by BrdU incorporation. Moreover, immunohistochemistry revealed that in DNase-treated mice, initial tumor growth was followed by complete tumor regression, accompanied by local and systemic CD8 T cell activation. Correspondingly, although a few metastases were detectable at day 14 in DNase-treated mice, no metastases were observed by day 28. Although it remains unclear whether tumor-derived or immune cell-derived traps are most relevant in our model, the results demonstrate that their presence in the TME promotes tolerance despite the inherent immune adjuvant properties of the traps. This suggests that DNase treatment could be particularly effective in combating early tumor development or relapse when used as part of a combination therapy designed to sustain anti-tumor immune responses. Citation Format: Sabina Sangaletti, Paola Portararo, Laura Botti, Valeria Cancila, Claudio Tripodo, Mario P. Colombo, Claudia Chiodoni. Degradation of extracellular trap DNA sustains anti-tumor immune responses in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C014.

Abstract B031: Lean adipocyte-derived lipid decreases metastatic potential of breast cancer cells

Abstract Obesity rates are increasing globally, making it of critical importance to study obesity-linked diseases. Breast cancer is the most common cancer in women, and obesity is associated with both increased incidence and metastatic progression of breast cancer. Metastasis formation depends on a cancer cell leaving the primary site, migrating through the bloodstream, and establishing new growth in a distant site. Both metastasizing cell-intrinsic and metastatic site-dependent factors can help to regulate the effectiveness of this process; however, it is unknown whether obesity-linked metastasis depends on changes to the metastasizing cell and/or changes to the distant metastatic site. This study aims to characterize how the lean versus obese tumor microenvironment impacts breast cancer metastasis and identify adipocyte-derived molecules that modulate the metastatic potential of breast cancer cells. We and others have shown that obesity leads to increased metastasis formation in mouse models of orthotopic breast cancer. However, my preliminary data show that an obese metastatic site is not sufficient to increase metastasis formation in an intravenous injection model of metastasis, emphasizing the importance of the primary tumor microenvironment in programming breast cancer cells for metastasis. Cancer cell stemness helps to mediate metastasis formation and can be assessed in vitro with the mammosphere assay. Importantly, my data demonstrate that a secreted lipid from lean, but not obese, adipocytes potently inhibits the ability of breast cancer cells to form mammospheres. Furthermore, this inhibition induces a G1/S cell cycle arrest. Together, these data suggest that lean adipocytes in the breast cancer tumor microenvironment act in a paracrine manner to decrease the metastatic potential of breast cancer cells. My work demonstrates the role of adipocytes in the regulation of metastasis in breast cancer and provides important insight into the role of the tumor microenvironment in mediating metastasis in lean versus obese conditions. Citation Format: Abigail E. Jackson, Keren I Hilgendorf. Lean adipocyte-derived lipid decreases metastatic potential of breast cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B031.

Abstract B035: Reciprocal macrophage - T cell interactions regulate anti-tumor immunity

Abstract Cancer immunotherapy (including immune checkpoint blockade (ICB)) has shown great promise in the treatment of some solid cancers, but responses in breast cancer patients are limited. While breast cancer is generally considered poorly immunogenic, the abundance of CD8+ T cells correlates with clinical response. CD8+ T cells are critical mediators of anti-tumor immunity and the main target for ICB. However, the onset of terminal functional T cell exhaustion poses a major challenge. Although T cell exhaustion has been linked to persistent antigen exposure, it remains unclear how the immune composition of the breast tumor microenvironment (TME) contributes to this dysfunctional T cell state. Breast cancers are heavily infiltrated with tumor-associated macrophages (TAMs) and their abundance correlates with T cell exhaustion and poor prognosis. Previously, we discovered a spatiotemporal co-dependency between exhausted T cells (TEX) and TAMs in mouse models of melanoma and breast cancer. We showed that TEX actively recruit monocytes to the TME and shape their differentiation trajectory into TAMs. Reciprocally, these TAMs ‘capture’ T cells in long-lived synaptic interactions that contribute to functional T cell exhaustion. Our current work is focused on studying the TEX-derived chemokines that regulate these TAM – T cell interactions in the breast TME with the ultimate goal to alleviate immune evasion and improve responsiveness of breast cancer to immunotherapy. Citation Format: Meenakshi Sudhakaran, Sofia Lombardi, Sophie Ayma, Matthew F. Krummel, Kelly Kersten. Reciprocal macrophage - T cell interactions regulate anti-tumor immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B035.

Abstract B040: Investigating the impact of tumor EMT states on immune cell infiltration in breast cancer

Abstract Breast cancer is one of the most frequently diagnosed cancers and the second leading cause of women’s cancer death. Although the five-year survival rate for women in the United States with nonmetastatic invasive breast cancer is 90%, this rate drops to less than 30% for those with tumors that have metastasized to distant organs. Epithelial-to-Mesenchymal Transition (EMT) plays a critical role in tumor metastasis by enabling the loss of epithelial characteristics and the gain of mesenchymal traits, leading to increased cell motility, invasion, and resistance to therapy. Consequently, targeting the EMT process may prevent tumor cell migration and improve patient outcomes. Rather than being a binary switch from an epithelial to a mesenchymal state, cells undergoing EMT can assume a spectrum of intermediate states, some of which may reshape the tumor microenvironment by recruiting pro-tumor immunosuppressive cells, such as regulatory T cells, M2-like macrophages, and myeloid-derived suppressor cells. However, the impact of EMT on the immune composition of the tumor microenvironment and the causative mechanisms remain poorly understood. Using multi-omics approaches in a mouse model of triple-negative breast cancer, we assessed the interaction between tumor and immune cells in the tumor microenvironment driven by the EMT. A heterogeneous mouse breast cancer cell line (4T1) was used to isolate five distinct single-cell-derived clonal populations residing in distinct states within the EMT spectrum. These EMT clones, along with the parental 4T1 cell line, were orthotopically transplanted into the mammary fat pads of BALB/c mice. Primary tumors were subsequently harvested and dissociated for single-cell RNA-sequencing (scRNA-seq) and DNA methylation analysis. Lungs were histologically evaluated for metastases. Tumors derived from an intermediate EMT clone exhibited increased tumor growth kinetics and a higher incidence of metastasis compared to epithelial or mesenchymal clones. In scRNA-seq data, tumors derived from different EMT clones largely retained their original EMT states in vivo and recruited distinct immune cell profiles. DNA methylation analysis further revealed that these tumors displayed unique methylation patterns in EMT-related CpGs. This study introduced a model that captures the complexity of the tumor microenvironment across the EMT spectrum. This was accomplished through immunophenotyping using scRNA-seq and identifying unique epigenetic patterns through DNA methylation. Our findings highlight the potential for distinct immune cell populations to be recruited to tumors in different EMT states, thereby influencing survival and metastasis outcomes. Furthermore, our results open new opportunities for developing cancer combination treatment strategies that target specific tumor-immune cell interactions. Citation Format: Hanxu Lu, Meisam Bagheri, Todd Miller, Diwakar Pattabiraman, Brock Christensen. Investigating the impact of tumor EMT states on immune cell infiltration in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B040.

Abstract C034: Investigating residual disease in its spatial context in BRCA1 p53-deficient mammary tumors

Abstract Although various effective anti-cancer treatments have become available over the last decades, resistance to all available therapies remains the major cause of death of cancer patients with disseminated tumors. Striking examples are patients with triple-negative breast cancer (TNBC), which are frequently defective in the repair of DNA double strand breaks, e.g. due to loss of BRCA1 function. Because of this defect, the patients initially respond very well to DNA damage-inducing chemotherapy. Unfortunately, disseminated tumors are usually not eradicated and resistant tumor cells are eventually selected from residual primary or metastatic tumor sites. It is therefore crucial to understand the molecular mechanisms underlying the drug tolerance of the residual tumor cells. To study residual disease, we used the K14cre;Brca1 F/F ;p53 F/F (KB1P) mouse model for hereditary breast cancer, which provides the unique opportunity to explore and target those mechanisms in a fully immunocompetent model. The mammary tumors that spontaneously develop highly resemble their human counterparts, both morphologically and in their therapy response. For example, tumors shrink in response to poly(ADP-ribose) polymerase inhibition, platinum-based treatment or the commonly used doxorubicin, docetaxel and cyclophosphamide (TAC) combination therapy. But despite repeated drug sensitivity, the KB1P mammary tumors are not eradicated, not even by a frequent dosing schedule. By combining single-cell RNA sequencing, spatial transcriptomics and imaging mass cytometry, we dissected the intratumoral heterogeneity and alterations in the tumor microenvironment of residual disease. We identified specific subpopulations of tumor cells that have a survival benefit after treatment, and these occurred together with an altered microenvironment characterized by a highly reactive stroma infiltrated with both anti-inflammatory and pro-tumoral immune cells. Interestingly, those structural and transcriptional changes are reversed in the relapsed tumors, highlighting the plasticity of drug tolerance. To investigate the mechanisms of the altered tumor-stroma interactions that are relevant for drug tolerance, we have designed a custom-made CRISPR/Cas9 library based on differential gene expression of the residual tumor cells. This library enables us to functionally test relevant mechanisms of drug tolerance in vivo and we expect that these analyses will provide useful insights into the tumor-stroma interactions that contribute to residual disease. Taken together, our detailed analyses demonstrate the substantial remodeling of tumor cells in their microenvironment upon treatment. To develop new therapeutic approaches to eradicate drug-tolerant tumor cells and thereby circumvent tumor relapse, it is essential to better understand the heterogeneous cellular composition of residual tumors in its spatial context. With this project, we hope to provide comprehensive data to develop better therapeutic strategies that target the tumor-stroma interaction and eradicate residual tumors. Citation Format: Morgane Decollogny, Demeter Túrós, Astrid Chanfon, Myriam Siffert, Ismar Klebic, Sven Rottenberg. Investigating residual disease in its spatial context in BRCA1 p53-deficient mammary tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C034.

Abstract B023: Regulatory T (Treg) cell contributes to tumor cell dissemination via extracellular matrix (ECM) remodeling, which is driven by TAMs in an IFNg-dependent manner

Abstract Background: The extracellular matrix (ECM) is a highly organized non-cellular network consisting of structural proteins, growth factors, cytokines and other secreted molecules. The ECM is highly dynamic and is a critical player in the regulation of local invasion and metastasis. The ECM network is ever-changing and an important immunosuppressive component of the tumor microenvironment (TME). However, the reciprocal effects of the immune system on the tumor-associated ECM are elusive and largely unexplored. Regulatory T (Treg) cells function to enforce peripheral tolerance and are potent suppressors of tumor immunity. We have previously shown that Treg cells promote tumor growth in murine breast cancer models by favoring alternative activation of tumor-associated macrophages (TAMs) via suppression of IFN-g. Aims: In this work, we sought to assess whether Treg cell-mediated immune suppression is associated with changes in the ECM, and whether those ECM changes functionally impact metastatic dissemination. Further, we assessed the requirement of TAMs and IFN-g in the ECM remodeling. Design Methods: In this study, we used spontaneous and transplantable models of breast cancer susceptible to genetic ablation of Treg cells. We used a combination of histopathological analysis, tumor decellularization, bioengineering 3D-chip modeling, in vivo manipulations and bioinformatic analysis, to evaluate remodeling of the ECM, cancer cell invasive behavior and metastatic dissemination. Results Summary: Treg cell ablation resulted in significant ECM remodeling, with reduced amounts and organization of collagen fibers, and increased amounts of fibronectin and laminin. Tumor cells seeding on Treg cell ablated tumor- derived decellularized ECM matrices results in reduced epithelial-mesenchymal transition (EMT) transcription factors, and profound impairment of collective migration. In vivo, Treg cell ablation in a neo-adjuvant setting followed by primary tumor resection resulted in significant reduction of circulating tumor cells (CTC) and impairment of lung metastatic disease. Importantly, we demonstrated that Treg cell ablation-driven changes in the matrisome correlate with long-term improved survival in a cohort of breast cancer patient samples. Through genetic knock-out models and in-vivo neutralization, we observed that Treg cell-dependent changes in ECM-related phenotypes are dependent on IFN-g-signaling. Single-cell RNA-sequencing (scRNA-seq) of murine breast tumors revealed that the matrisome signature is highly upregulated among TAMs, compared to other TME cell types. Lastly, using conditional genetic knock-out models we show these IFN-g-dependent changes are mostly driven by TAMs. Conclusions: Altogether, we identified a novel metastasis-promoting effect of Treg cells in the breast cancer microenvironment through regulation of ECM dynamics beyond their described effects on primary tumor growth. Citation Format: Ailen D. Garcia-Santillan, Jessanne Y. Lichtenberg, He Shen, Jasmine M Rodriguez, Jonathan Barra, Nicholas M Clark, Wei Du, Leandro M Martinez, Ashley D Hadjis, Taylor Calicchia, Mikhail G Dozmorov, Jose J Bravo-Cordero, Amy L Olex, Priscilla Y Hwang, Paula D Bos. Regulatory T (Treg) cell contributes to tumor cell dissemination via extracellular matrix (ECM) remodeling, which is driven by TAMs in an IFNg-dependent manner [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B023.

Abstract C012: Dissemination of retrotransposable elements from the non-coding genome in tumor-derived extracellular vesicles target stromal and immune cells to induce local and systemic inflammation

Abstract The majority of the human genome (>70%) contains non-coding DNA consisting of retrotransposable elements (RTE), such as LINE-1, SINE/Alu elements, human endogenous retroviruses (HERV), and satellite repeat DNA (Hsat2 and Hsat3). The expression of these elements is suppressed in normal adult tissues but are abnormally expressed in many cancers. Another key mechanism in cancer initiation and progression is the release of tumor-derived extracellular vesicles (EV) that are long-distance communication vehicles propagating signals from tumors inducing metabolic changes, immune dysregulation and promoting metastases across the body. Our work has uncovered a link between RTE, HERV and Hsat expression in cancer and EV in driving tumor progression and immune dysregulation. W hole transcriptome sequencing of EV derived from several different cancer cell lines and patient samples, including Ewing sarcoma, osteosarcoma, prostate, pancreatic, and breast cancer found that RTE, HERV and Hsat2,3 transcripts are highly enriched in EV versus coding region transcripts and in much higher abundance than in the tumor cell themselves. Tumor-derived EV contained dsRNA, dsDNA and RNA:DNA hybrids from these non-coding regions. Plasma EV isolated from both metastatic and non-metastatic patients also showed an enrichment of these RTE, HERV and Hsat2,3 elements and clearly differentiated healthy, normal donor EV that had little or no detectable signal. Evidence of local dissemination of these non-coding elements in EV was also found in vivo in tumor xenografts that showed transfer of Hsat2,3 transcripts into the murine stroma that naturally lack these satellite repeats. Treatment of fibroblasts and myeloid cells with tumor-derived EV triggered innate immune responses via type I IFN and pro-inflammatory cytokines, including IL-1beta, IL-6, IL-8, and TNFalpha owing to the pathogen-like nucleic acid sequences (viral mimicry) of these elements. These EV-induced chronic inflammatory effects were mainly induced through the cGAS-STING nucleic acid sensing pathway. Evidence of DNA damage in stromal fibroblasts and induced senescence was also found as a result of EV treatment. A key element of RTE is the expression of LINE-1 and HERV derived reverse transcriptase (RT) that propagates the action of RTE via RNA:DNA hybrids, dsDNA and retrotransposition in the genome. In a spontaneous estradiol-driven breast cancer model in ACI rats, long-term treatment with an HIV RT inhibitor (lamivudine/3TC) inhibited breast tumor development associated with reduced RTE activation in the mammary gland and in circulating immune cells as well as reduced chronic systemic innate inflammation. Our results suggest that RTE, HERV and Hsat activation, together with their local and systemic dissemination in EV plays an important role in cancer initiation and progression associated with chronic inflammation. We also suggest that abnormal dissemination of these activated non-coding elements in EV may also play a role in "inflammaging" facilitating not only cancer but also other chronic diseases. Citation Format: Laszlo Radvanyi, Valentina Evdokimova, Peter Ruzanov, Zhenbo Zhang, Poul Sorenson, Hendrik Gassmann, Stefan Burdach, Lincoln D. Stein. Dissemination of retrotransposable elements from the non-coding genome in tumor-derived extracellular vesicles target stromal and immune cells to induce local and systemic inflammation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C012.

Abstract B012: Understanding the role of VISTA in regulating T cell function and the therapeutic potential of blocking VISTA in triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is the most aggressive and highly metastatic breast cancer subtype with limited effective treatment options. Immunotherapy with immune checkpoint inhibitors (ICIs) targeting checkpoint molecules have been widely used and have shown therapeutic efficacy in treating multiple malignancies in the last decade. In TNBC, ICI targeting PD-1, alone or in combination with chemotherapy has been approved for the treatment of patients with both early and advanced stage of the disease. Although, there has been significant clinical benefit achieved from anti-PD-1 and chemotherapy, treatment efficacy is often limited to a subgroup of patients. The limited response to ICIs has been attributed to the increased infiltration of immunosuppressive cells (including MDSCs, neutrophils and tumor-associated macrophages), more importantly, the upregulation of alternative immune checkpoint receptors/ligands that may further suppress cytotoxic T cells (CD8+ T cells) in the tumor microenvironment (TME). Therefore, it has been suggested that targeting other immune checkpoint molecules may be more effective in bolstering anti-tumor immune response. V- domain Ig suppressor of T-cell activation (VISTA) is one of the immune checkpoint molecules attracting increasing attention in the context of anti-tumor immunity due to its broad expression across various cancer types and increased expression upon development of immune checkpoint resistance. However, the role of VISTA in regulating the anti-tumor immune response and its therapeutic potential in TNBC remains unclear. In our study, we observed that VISTA is expressed in TNBC patients, predominantly in neutrophils and macrophages. In the Py230 murine breast cancer model, VISTA expression increased upon paclitaxel (PTX), anti-PD-L1 and paclitaxel combined with anti-PD-L1 treatment compared to the control group, suggesting its potential role as a resistance mechanism in sustaining or further inhibiting T cells. In the Py230 model, VISTA blockade increased the infiltration and activation of CD8+ T cells, as evidenced by the higher number of CD8+ T cells, increased CD69+CD8+ T cells, and Granzyme B production, indicating that VISTA blockade improved CD8+ T cells mediated anti-tumor immunity. To functionally test the role of VISTA, we stimulated CD8+ T cells and co-cultured them with naïve macrophages or tumor educated macrophages in the presence or absence of VISTA blocking antibody. Tumor educated macrophages showed increased immunosuppressive activity which was abrogated with VISTA blockade. Moreover, we found that the immunosuppressive effect of macrophages on T cell activation needs direct cell-cell interaction between VISTA on macrophages and T cells. In conclusion, this study shows the mechanism of action of VISTA in the regulation of T cells mediated anti-tumor immune response and provides the rationale for targeting VISTA in TNBC. Citation Format: Maidinaimu Abudula, Yuliana Astuti, Meirion Raymant Raymant, Vijay Sharma, Michael Schmid, Ainhoa Mielgo. Understanding the role of VISTA in regulating T cell function and the therapeutic potential of blocking VISTA in triple negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B012.

PLLA Porous Scaffold as a 3D Breast Cancer Model to Investigate Drug Resistance.

Multidrug resistance remains one of the major challenges in breast cancer research, often leading to treatment failure. To better understand this mechanism, sophisticated three-dimensional (3D) tumor models are necessary, as they offer several advantages over traditional bidimensional (2D) cultures. In this study, poly-l-lactic-acid porous scaffolds were produced using a thermally induced phase separation technique and employed as 3D models for breast cancer cell lines: MDA-MB-231, MCF-7, and its multidrug-resistant variant, MCF-7R. The MTS assay was used to compare growth inhibition following doxorubicin treatment in 2D and 3D. Remarkably, the IC50 values increased in 3D cultures compared to 2D: MDA-MB-231 (445 vs. 54.5 ng/mL), MCF-7 (7.45 vs. 0.75 μg/mL), and MCF-7R (165 vs. 39 μg/mL). MCF-7R, which usually shows greater resistance in 2D, demonstrated even higher resistance in 3D. In fact, IC50 was not reached within 3 days as with the other models, but only after 6 days. Cellular morphology also played a crucial role. When treated with concentrations higher than the IC50, MDA-MB-231 cells lost their physiological 3D clustered structure, while MCF-7 and its resistant variant exhibited disrupted layers. All cell lines in 3D showed higher chemoresistance, suggesting a more biomimetic spatial architecture. Our work bridges the gap between monolayer and animal models, highlighting the potential of polymeric 3D scaffolds in breast cancer research.

Insulin-like growth factor-binding protein-3 is induced by tamoxifen and fulvestrant and modulates fulvestrant response in breast cancer cells

IntroductionInsulin-like growth factor binding protein-3 (IGFBP-3) exerts varying effects on estrogen receptor alpha (ERα)-positive and triple-negative breast cancer (TNBC) cells. In ERα-positive cells, IGFBP-3 is antiproliferative and proapoptotic. In contrast, IGFBP-3 stimulates proliferation in triple-negative breast cancer (TNBC) cells via EGFR activation.MethodsTo identify potential mechanisms that underlie the opposing effects of IGFBP-3 on these two breast cancer subtypes, IGFBP-3 expression was determined in cell line models of both ERα-positive breast cancer and TNBC, and cells were treated with antiestrogens tamoxifen and fulvestrant. Results and discussionMCF-7 and T-47D cells expressed low levels of IGFBP-3 when compared to MDA-MB-231 and MDA-MB-468 cells. MCF-7 cells with acquired resistance to the selective estrogen receptor degrader fulvestrant expressed high IGFBP-3 and MCF-7 cells with constitutive IGFBP-3 expression were fulvestrant resistant. IGFBP-3 expression was increased in all cell lines upon treatment with fulvestrant or the selective estrogen receptor modulator tamoxifen and both fulvestrant and tamoxifen increased TNBC cell proliferation. Further, IGFBP-3 expression was increased by treatment with the GPER1 agonist G-1 and attenuated upon treatment with P17, a YAP/TAZ inhibitor. These data suggest that IGFBP-3 modulates breast cancer cells and is a mediator of breast cancer cell response to fulvestrant and tamoxifen.

Abstract A003: Snord67 promotes breast cancer metastasis through U6-mediated alternative splicing

Abstract Small nucleolar RNAs (snoRNAs) a class of ncRNAs that canonically guide post-transcriptional modifications, including 2'-O-methylation, on ribosomal RNA (rRNA) and small nuclear RNA (snRNA). While traditionally considered housekeeping genes, snoRNAs have increasingly been found to function in diverse physiologic and pathologic processes, including cancer. In an immune-competent murine model of triple negative breast cancer (TNBC) lymphatic metastasis, we identified the snoRNA Snord67 as one of the most upregulated noncoding RNAs in axillary LN (AxLN) tumors relative to mammary fat pad (MFP) tumors and lung metastases. Loss of Snord67 resulted in decreased colony formation and spheroid size in murine and human TNBC cell lines, and led to decreased lymph node tumor growth and decreased distant metastases in two immune-competent murine models of TNBC lymphatic dissemination. To determine the mechanism by which Snord67 promotes tumor growth and metastasis, we examined the impact of Snord67 on 2'-O-methylation, gene expression, and alternative splicing. Loss of Snord67 in TNBC cell lines led to decreased 2'-O-methylation at the C60 nucleotide (Cm60) in the core spliceosome component U6 snRNA. Re-introduction of wild-type Snord67 rescued Cm60 in U6 snRNA and rescued the colony formation and spheroid phenotypes of the Snord67 knockout cell lines. However, a mutant Snord67 incapable of guiding U6 Cm60 only partially rescued these in vitro phenotypes, suggesting that Snord67 promotes in vitro tumor cell growth at least in part by guiding Cm60 in U6 snRNA. We then performed RNA sequencing of Snord67 knockout and wild-type murine and human TNBC cell lines. We found that loss of Snord67 led to widespread changes in alternative splicing, consistent with its role in guiding 2'-O-methylation of the core spliceosome component U6. We further demonstrated that the inclusion of alternatively spliced cassette exons in MYO18A and NFYA was not only downregulated upon Snord67 knockout in a human TNBC cell line, but also positively correlated with Snord67 expression levels in primary breast tumors and lymph node metastases from breast cancer patients. Based on these results, we propose a model in which Snord67 guides U6 Cm60, which leads to a pro-metastatic alternative splicing program and thereby promotes lymph node tumor growth and distant metastasis. Citation Format: Katherine I Zhou, Yvonne Chao, Kwame K Forbes, Alessandro Porrello, Gabrielle M Gentile, Aaron C Chack, Dixcy J.S. John Mary, Haizhou Liu, Yinzhou Zhu, Eric Cockman, Lincy Edatt, Grant A Goda, Justin Zhao, Hala Abou Assi, Hannah J Wiedner, Yi-Hsuan Tsai, Lily Wilkinson, Amanda E Van Swearingen, Lisa A Carey, Jimena Giudice, Daniel Dominguez, Christopher L Holley, Chad V Pecot. Snord67 promotes breast cancer metastasis through U6-mediated alternative splicing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A003.

Abstract I008: Antisense Oligonucleotides as Therapeutics for Difficult-to-Drug Targets in Oncology

Abstract Many potential oncology targets are difficult to modulate by conventional classes of agents. Interfering with these targets at the level of RNA is a novel approach for difficult-to target proteins, as well as for non-coding RNAs, emerging targets that are part of the “dark matter’ of the genome. Antisense oligonucleotide (ASO) therapeutics are short sequences of 12- 30 DNA/RNA residues, designed to bind the cognate transcribed RNA. The ASO is chemically modified to increase stability, improve binding affinity, optimize pharmacokinetic properties and reduce immunostimulatory potential. The hybridization of ASO to cognate RNA results in cleavage of bound RNA by RNAse H1 and subsequent degradation. Several ASOs are marketed for treatment of rare diseases, but to date none have been approved for oncology indications. The long-noncoding RNA (lncRNA) MALAT1 is an example of an oncology target uniquely suited for an ASO therapeutic. This lncRNA is not translated and resides mostly in the nucleus, limiting access for most therapeutic approaches. MALAT1 is highly upregulated in various cancers, appears to play a role in epithelial to mesenchymal transition, and is associated with disease progression in patients. Inhibition of MALAT1 in preclinical breast cancer models, either genetic or pharmacologic using a murine ASO, leads to changes in tumor architecture and reduction in metastasis. FLM-7523 is a 16-mer ASO designed to bind to human MALAT1. It is a generation 2.5 design based on the constrained ethyl (cEt) modification to the 3 residues on the 3’ and 5’ ends of the ASO, and administered in a saline solution without any delivery vehicle. Preclinical studies have shown that FLM-7523 effectively inhibits MALAT1 expression in in vitro and in vivo models, and GLP toxicology studies have been completed to support a First-in-Human clinical trial. The signal transduction molecule STAT3 is another oncology target of interest that has, to date, been difficult to modulate. Danvatirsen is a 16-mer generation 2.5 ASO targeting human STAT3 mRNA and is currently in clinical trials. Preclinical in vivo studies in murine tumor models have demonstrated a reduction in STAT3 mRNA and protein following treatment with a STAT3 targeting ASO, along with tumor growth inhibition. Combination treatments with immune checkpoint inhibitors, including anti-PD-1 and anti-PD-L1, demonstrate improved tumor growth inhibition relative to monotherapy. In clinical trials, tumor uptake of danvatirsen is observed in post-treatment biopsies, with corresponding reduction in STAT3 protein. Danvatirsen is generally well tolerated with the most common adverse events being increased liver function tests and decreased myeloid hematologic parameters, both of which are easily monitored, manageable, and reversible. Clinical responses have been observed with the most robust being in combination with a PD-L1 inhibitor in recurrent/metastatic head and neck squamous cell carcinoma. In conclusion, ASOs are novel therapies that can inhibit oncology targets not tractable by traditional approaches. Citation Format: Andrew Denker. Antisense Oligonucleotides as Therapeutics for Difficult-to-Drug Targets in Oncology [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr I008.

Green synthesized Zingiber officinale-ZnO nanoparticles: anticancer efficacy against 3D breast cancer model.

Aim: ZnO NPs were prepared via green synthesis utilizing Zingiber Officinale.Methodology: Physical characterization and biological activity were performed against 2D, and 3D spheroids MCF-7 cell lines.Results: The NPs exhibited 188.9, 175.7 and 171.2nm size with charge of -8.2, -11.7 and -9.7mV for the 2%, 3% and 4% formulations. XRD confirmed a wurtzite hexagonal phase. FTIR spectra showed Zn-O stretching vibrations. The 2%, 3% and 4% formulations presented IC50 values of 14.7, 26.2 and 47μg/ml, respectively, with complete destruction of MCF-7 spheroids. Elevated TNF-α levels suggested an inflammatory-mediated mechanism of action.Conclusion: 2% Zingiber officinale-derived ZnO NPs showed antitumor potential against deserving further mechanistic and in vivo explorations.

Cationic Metal‐Organic Layer Delivers siRNAs to Overcome Radioresistance and Potentiate Cancer Radiotherapy

Radiotherapy plays an important role in modern oncology, but its treatment efficacy is limited by the radioresistance of tumor cells. As a member of the inhibitor of apoptosis protein family, survivin plays a key role in developing radioresistance by mediating apoptosis evasion, promoting epithelial‐mesenchymal transition, and modulating cell cycle dynamics. Efficient downregulation of survivin expression presents a promising strategy to enhance the antitumor effects of radiotherapy. Herein, we report the design of a hafnium‐porphyrin‐based cationic metal‐organic layer (CMOL) with quaternary ammonium capping groups to deliver small interfering RNAs (siRNAs) for enhanced radiotherapy. The CMOL@siRNA nanoplatform not only increased energy deposition from X‐rays and reactive oxygen species generation via a unique radiotherapy‐radiodynamic therapy process, but also effectively delivered siRNAs to downregulate survivin expression and ameliorate radioresistance of cancer cells. Consequently, CMOL@siRNA in combination with low‐dose X‐ray irradiation demonstrated remarkable antitumor efficacy with 96.9% and 91.4% tumor growth inhibition in murine colorectal carcinoma and triple‐negative breast cancer models, respectively.

MicroRNAome profiling of breast cancer unveils hsa-miR-5683 as a tumor suppressor microRNA predicting favorable clinical outcome.

Breast cancer is a heterogeneous disease with diverse molecular subtypes, underscoring a better understanding of its molecular features and underlying regulatory mechanisms. Therefore, identifying novel prognostic biomarkers and therapeutic targets is crucial for advancing the current standard of care for breast cancer patients. Ninety-six formalin-fixed paraffin-embedded (FFPE) breast cancer samples underwent miRNAome profiling using QIAseq microRNA library kit and sequencing on Illumina platform. Mature miRNA quantification was conducted using CLC Genomics Workbench v21.0.5, while Relapse-free survival (RFS) analysis was conducted using RStudio 2023.09.1. Gain-of-function studies were conducted using miRNA mimics, while the effects of miRNA exogenous expression on cancer hallmark were assessed using 2-dimentional (2D) proliferation assay, three-dimensional (3D) organotypic culture, and live-dead staining. TargetScan database and Ingenuity Pathway Analysis (IPA) were used for miRNA target identification. Hierarchical clustering based on miRNA expression revealed distinct patterns in relation to PAM50 classification and identified miRNAs panels associated with luminal, HER2, and basal subtypes. hsa-miR-5683 emerged as a potential prognostic biomarker, showing a favorable correlation with RFS and suppressing tumorigenicity under 2D and 3D conditions in triple-negative breast cancer (TNBC) models. Findings were further extended to the MCF7 hormone receptor positive (HR+) model. Transcriptomic profiling of hsa-miR-5683 overexpressing TNBC cells revealed its potential role in key oncogenic pathways. Integration of downregulated genes and CRISPR-Cas9 perturbational effects identified ACLY, RACGAP1, AK4, MRPL51, CYB5B, MKRN1, TMEM230, NUP54, ANAPC13, PGAM1, and SOD1 as bona fide gene targets for hsa-miR-5683. Our data provides comprehensive miRNA expression atlas in breast cancer subtypes and underscores the prognostic and therapeutic significance of numerous miRNAs, including hsa-miR-5683 in TNBC. The identified gene targets unravel the intricate regulatory network in TNBC progression, suggesting promising avenues for further research and targeted therapeutic interventions.

Cationic Metal-Organic Layer Delivers siRNAs to Overcome Radioresistance and Potentiate Cancer Radiotherapy.

Radiotherapy plays an important role in modern oncology, but its treatment efficacy is limited by the radioresistance of tumor cells. As a member of the inhibitor of apoptosis protein family, survivin plays a key role in developing radioresistance by mediating apoptosis evasion, promoting epithelial-mesenchymal transition, and modulating cell cycle dynamics. Efficient downregulation of survivin expression presents a promising strategy to enhance the antitumor effects of radiotherapy. Herein, we report the design of a hafnium-porphyrin-based cationic metal-organic layer (CMOL) with quaternary ammonium capping groups to deliver small interfering RNAs (siRNAs) for enhanced radiotherapy. The CMOL@siRNA nanoplatform not only increased energy deposition from X-rays and reactive oxygen species generation via a unique radiotherapy-radiodynamic therapy process, but also effectively delivered siRNAs to downregulate survivin expression and ameliorate radioresistance of cancer cells. Consequently, CMOL@siRNA in combination with low-dose X-ray irradiation demonstrated remarkable antitumor efficacy with 96.9% and 91.4% tumor growth inhibition in murine colorectal carcinoma and triple-negative breast cancer models, respectively.

Phase Ib clinical and pharmacodynamic study of the TIE2 kinase inhibitor rebastinib with paclitaxel or eribulin in HER2-negative metastatic breast cancer.

Breast cancer cells disseminate to distant sites via Tumor Microenvironment of Metastasis (TMEM) doorways. The TIE2 inhibitor rebastinib blocks TMEM doorway function in the PyMT mouse model of breast cancer. We aimed to assess the safety and pharmacodynamics of rebastinib plus paclitaxel or eribulin in patients with HER2-negative metastatic breast cancer (MBC). This phase Ib trial enrolled 27 patients with MBC who received 50 mg or 100 mg of rebastinib PO BID in combination with weekly paclitaxel 80 mg/m2 (if ≤ 2 prior non-taxane regimens) or eribulin 1.4 mg/m2 on days 1 & 8 (if ≥ 1 prior regimen). Safety, tolerability and pharmacodynamic parameters indicating TIE2 kinase inhibition and TMEM doorway function were evaluated. No dose-limiting toxicities in cycle 1 or 2 were observed among the first 12 patients at either rebastinib dose level. The most common treatment-emergent adverse events (AEs) were anemia (85%), fatigue (78%), anorexia (67%), leukopenia (67%), increased alanine aminotransferase (59%), hyperglycemia (56%), nausea (52%), and neutropenia (52%). AEs attributed to rebastinib include muscular weakness and myalgias. Intraocular pressure increased at the 100 mg rebastinib dose level, whereas angiopoietin-2 levels increased at both dose levels, providing pharmacodynamic evidence for TIE2 blockade. Circulating tumor cells decreased significantly with the combined treatment. Objective response occurred in 5/23 (22%) evaluable patients. In patients with MBC, the recommended phase 2 dose of rebastinib associated with pharmacodynamic evidence of TIE2 inhibition is either 50 or 100 mg PO BID in combination with paclitaxel or eribulin.

Brca1 haploinsufficiency promotes early tumor onset and epigenetic alterations in a mouse model of hereditary breast cancer.

Germline BRCA1 mutation carriers face a high breast cancer risk; however, the underlying mechanisms for this risk are not completely understood. Using a new genetically engineered mouse model of germline Brca1 heterozygosity, we demonstrate that early tumor onset in a Brca1 heterozygous background cannot be fully explained by the conventional 'two-hit' hypothesis, suggesting the existence of inherent tumor-promoting alterations in the Brca1 heterozygous state. Single-cell RNA sequencing and assay for transposase-accessible chromatin with sequencing analyses uncover a unique set of differentially accessible chromatin regions in ostensibly normal Brca1 heterozygous mammary epithelial cells, distinct from wild-type cells and partially mimicking the chromatin and RNA-level changes in tumor cells. Transcription factor analyses identify loss of ELF5 and gain of AP-1 sites in these epigenetically primed regions; in vivo experiments further implicate AP-1 and Wnt10a as strong promoters of Brca1-related breast cancer. These findings reveal a previously unappreciated epigenetic effect of Brca1 haploinsufficiency in accelerating tumorigenesis, advancing our mechanistic understanding and informing potential therapeutic strategies.