Mammary Tumor Research Articles

Abstract 4178: Non-invasive shortwave infrared imaging of cytotoxic T lymphocyte infiltration for monitoring responses to combination immunotherapy and chemotherapy

Abstract Although triple-negative breast cancer (TNBC) can be treated with anti-PD-1 checkpoint immunotherapy in combination with chemotherapy, there remains a challenge in effectively monitoring therapeutic responses. Current non-invasive clinical imaging tools to evaluate response to treatment are reliant upon measurements of tumor volume and may fail to distinguish true progression from increased immune cell infiltration. Invasive biopsy sampling and immunohistochemistry (IHC) can elucidate changes in the immune landscape of treated tumors, but these methods are not conducive to providing real-time information. This study presents shortwave infrared (SWIR) imaging as a potential tool to detect treatment-induced cytotoxic T lymphocyte (CTL) infiltration non-invasively and in real time using rare earth metal-doped nanoparticles encapsulated in human serum albumin nanocomposites (ReANCs). ReANCs were chemically conjugated with anti-CD8α antibodies as targeting ligands to facilitate binding of the nanoprobes to CTLs with high specificity in a syngeneic mouse model of breast cancer. After treating the mice with combination anti-PD-1 and doxorubicin, volumetric analysis of the mammary fat pad tumors did not show any significant impact of treatment compared to single treatment and untreated control mice. However, increased CTL infiltration in the tumors of mice that received combination treatment was detected by in vivo SWIR imaging. CTL infiltration was validated with ex vivo IHC staining, and a monotonic relationship was observed between SWIR fluorescence and CD8 positivity. IHC staining of other immune markers, including CD45, CD3, CD4, and PD-L1, showed that combination treatment may influence in the expression of these markers, presenting additional targets that could be imaged with ReANCs in the future. In conclusion, the increase in SWIR signal from CD8-targeted ReANCs in tumors treated with combination immunotherapy and chemotherapy and the relationship with IHC staining highlight the ability to use SWIR imaging for non-invasive assessment of changes in immune dynamics following treatment. Citation Format: Jay V. Shah, Jake N. Siebert, Xinyu Zhao, Shuqing He, Richard E. Riman, Mei Chee Tan, Mark C. Pierce, Edmund C. Lattime, Vidya Ganapathy, Prabhas V. Moghe. Non-invasive shortwave infrared imaging of cytotoxic T lymphocyte infiltration for monitoring responses to combination immunotherapy and chemotherapy [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 4178.

Abstract 6958: CRIPTO-regulated extracellular vesicles mediate heterotypic cellular crosstalk in triple negative breast cancers

Abstract Tumor progression involves dynamic interaction between various cell types within the tumor microenvironment. Fibroblasts, which normally participate in wound healing, reciprocally communicate with breast cancer cells to promote tumor cell plasticity and heterogeneity. Gaining insights into the molecular mechanisms underlying this cell-to-cell communication, could unlock novel therapeutic avenues for inhibiting cellular plasticity, breast cancer progression and metastasis. The small GPI-anchored adaptor protein CRIPTO is a key player in this context though important molecular details remain to be elucidated. CRIPTO orchestrates TGF-β pathway signaling as well as growth factor-like signaling pathways that activate c-Src/MEK/AKT, in an autocrine and paracrine manner. CRIPTO is expressed during mammary development, wound healing, and breast cancer but is largely undetectable in homeostatic tissue, making it a promising therapeutic target. Our previous studies have demonstrated that the activity of CRIPTO is contingent upon cellular stress, with pronounced influence within pro-fibrotic, hypoxic domains of tumors. Importantly, we have shown that inhibition of CRIPTO using the soluble recombinant protein antagonist A4Fc effectively impedes the progression of triple negative breast cancer (TNBC) in murine xenograft models. Building upon these findings, we have been dissecting CRIPTO’s role in coordinating fibroblast activation and cell-state reprogramming in TNBC. We find that inhibition of CRIPTO with A4Fc suppresses cancer associated fibroblast (CAF) activation and collagen remodeling in vivo. Further, by modeling tumor cell:fibroblast crosstalk in vitro, we have demonstrated mechanistically that CRIPTO expression in tumor cells mediates subsequent extracellular vesicle (EV) uptake by fibroblasts, as well as fibroblast SMAD pathway activation and priming for reciprocal signaling back to tumor cells to promote transcriptional reprogramming and cellular invasion.The significance of these findings lies in their elucidation of key EV control mechanisms that could be exploited to intervene in essential tumor cell:fibroblast crosstalk or to enhance targeting of therapeutic moieties based on EV architecture and function in cancer and/or normative regenerative processes. Citation Format: David W. Freeman, Brooke L. Gates, Mauri D. Spendlove, Benjamin T. Spike. CRIPTO-regulated extracellular vesicles mediate heterotypic cellular crosstalk in triple negative breast cancers [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 6958.

Abstract 210: Carcinogenic influence of e-cigarette aerosols on breast and lung cancer models

Abstract Background and Purpose: With the rising popularity of E-cigarettes among the youth, there is an urgent need to understand their long-term health implications, particularly the potential for carcinogenesis. Although tobacco smoking is established as a major cancer risk factor, the carcinogenic potential of E-cigarettes remains underexplored. Given that metastatic cancer accounts for most cancer-related deaths in the United States, elucidating the biological impacts of E-cigarette aerosols on cancer progression is critical for developing effective interventions. This study aims to address this critical gap by exploring the effects of E-cigarette aerosols on cellular models pertinent to breast and lung cancer. Methods: Mouse breast epithelial cells (HC11), triple-negative breast cancer cells (4T1), and human lung adenocarcinoma cells (Calu-3) were exposed to E-cigarette aerosol at the air-liquid interface for one hour, followed by a 24-hour recovery period. Results: Our study revealed that E-cigarette aerosols with mint flavor induce epithelial-to-mesenchymal transition (EMT) in both normal mammary epithelial and breast cancer cells. Furthermore, we also observed activation of the TGF pathway and an increased expression of stemness-related markers (OCT4, SOX2, NANOG, KLF4, and c-MYC) in 4T1 cells exposed to mint and menthol-flavored E-cigarette aerosols in both 2D and 3D cell culture environments. Microscopic examination at a 10X magnification level revealed that 4T1 and Calu-3 cells exposed to menthol-flavored E-cigarette aerosols exhibited signs of cellular disassociation and detachment whereas the control cells maintained discoidal aggregate morphology. Conclusion: The data presented here illuminate a potential link between E-cigarette use and carcinogenic processes, challenging the notion of E-cigarettes as a safe smoking alternative. This research is of paramount importance to cancer research as it unveils possible new avenues for tumor progression, emphasizing the necessity for a critical evaluation of E-cigarette products. Citation Format: Rizwana Begum, Shilpa Thota, Naveen Chintala, Biplov Sapkota, Abhishek Pandit, Joseph Francis. Carcinogenic influence of e-cigarette aerosols on breast and lung 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 210.

Abstract 6605: Understanding the role of sumoylated Etv1 in mammary oncogenesis

Abstract The Small Ubiquitin-like Modifier (SUMO) pathway is a major post-translational regulatory mechanism involved in a variety of cellular functions including the DNA damage response, transcriptional regulation, and carcinogenesis. Recent discoveries by our group demonstrated that inhibiting the SUMO pathway can repress the growth of breast cancer xenografts and Neu-driven mammary oncogenesis in a genetically engineered mouse model with conditional knockout (CKO) of the SUMOylation E2 conjugating enzyme, Ube2i. However, despite the consistency of the findings, there remains considerable debate concerning the mechanisms through which SUMO inhibition represses tumorigenesis. Our downstream analysis suggested ETS Translocation Variant 1, encoded by ETV1 gene, acts as a SUMO-sensitive transcription factor that drives mammary oncogenesis and cancer growth. To examine oncogenic properties of SUMO-conjugated ETV1 in breast cancer, we cloned HA-tagged WT Etv1, SUMO-resistant Etv15xKR, or SUMO mimetic SUMO-1-Etv15xKR into a lentiviral vector with a GFP cassette. Proliferation, colony formation in soft agar, and in vivo xenograft assays were evaluated in the Ube2iF/F cell line derived from a tumor in a MMTV-c-neu/Ube2ifl/fl animal. We did ChIP-seq in Ube2iF/F cells using SUMO-1 and Etv1 antibodies to examine the effects of SUMO conjugation on Etv1 genomic occupancy. RNA-seq using siEtv1 or AdCre was also performed to compare changes in gene expression with SUMO-conjugated Etv1 genomic occupancy. Inhibition of SUMOylation via AdCre caused a significant reduction in proliferation and colony formation in vitro and completely block the formation of flank xenografts in vivo. Interestingly, expressing a SUMO-resistant ETV15xKR rescued in vitro growth effects, whereas a SUMO mimetic SUMO-1-ETV15xKR had no effect on restoring proliferation. On the contrary, in vivo xenograft assays demonstrated that the Etv15xKR-SUMO1 efficiently induced tumor formation despite the loss of Ube2i while the sumo-resistant Etv15xKR was unable to rescue tumorigenesis. Bulk RNA-seq and ChIP seq performed demonstrated different patterns of gene regulation between sumoylated and unsumoylated Etv1. Gene set enrichment analysis (GSEA) showed genes associated with cancer stemness were significantly enriched in cells expressing Etv15xKR-SUMO1. Collectively, these observations support Etv1 acting as a SUMO-sensitive transcription factor that drives a stem cell phenotype necessary for mammary oncogenesis and progression. Citation Format: Zhijie Li, Kelsey E. Koch, Dakota T. Thompson, Dana M. Van der Heide, Jeremy Chang, Christopher M. Franke, Mohammed O. Suraju, Anna C. Beck, Allison W. Lorenzen, Jeffrey R. White, Mikhail V. Kulak, David K. Meyerholz, Colin Kenny, Ronald J. Weigel. Understanding the role of sumoylated Etv1 in mammary oncogenesis [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 6605.

Abstract 7368: Sentinel omics signatures: Comparative clinical and blood sample analysis from canine mammary tumor patients and their human guardian

Abstract Breast cancer remains a leading cause of morbidity and mortality worldwide, necessitating innovative approaches for early detection and intervention. This study leverages the comparative biology paradigm, exploring the shared oncological pathways between humans and canines through a unique multi-omics approach. We have gathered a diverse cohort comprising healthy dogs, canine mammary tumor patients, and their respective human guardians, collecting comprehensive clinical data alongside blood samples. Employing RNA-seq, MBD-seq, and LCMS, we aim to construct a detailed transcriptomic, methylomic, and proteomic landscape to elucidate potential sentinel biomarkers indicative of an oncogenic environment shared between species. While results are forthcoming, the premise of our research rests on the hypothesis that environmental factors contributing to mammary cancer may manifest systemically, potentially affecting both canines and humans. By delineating these interspecies molecular signatures, we hope to advance the field of precision medicine, offering insights into early detection and the systemic nature of cancer pathobiology. This abstract presents the foundation and direction of our ongoing investigation, with a commitment to providing a comprehensive update, inclusive of significant findings, at the conference. Citation Format: Keun Hong Son, A-Reum Nam, Hyoung-Min Park, HuiSu Kim, Jeong-Woon Lee, Je-Yoel Cho. Sentinel omics signatures: Comparative clinical and blood sample analysis from canine mammary tumor patients and their human guardian [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 7368.

Abstract 6556: Targeting mesenchymal tumor cell-intrinsic factors sensitizes refractory tumors to immune checkpoint blockade therapy

Abstract Immune checkpoint blockade (ICB) therapy has emerged as a promising approach for treating breast cancer, however, only a fraction of patients derives clinical benefit and the underlying reasons for heterogeneous responses remain elusive. The epithelial-to-mesenchymal transition (EMT) enables these carcinomas to metastasize and acquire resistance to chemotherapy. We have recently demonstrated that the EMT program also confers resistance to immunotherapies. Specifically, epithelial tumors recruit CD8+ T-cells to the tumor microenvironment and are sensitive to anti-CTLA4 ICB. In contrast, mesenchymal tumors assemble an immunosuppressive tumor microenvironment and are resistant to the same treatment. Furthermore, mesenchymal tumors express multiple immunosuppressive paracrine factors relative to their epithelial counterparts. Of these, abrogation of CSF1 or SPP1 from mesenchymal cancer cells generates partial responses to ICB therapy. Strikingly, abrogation of CD73 from mesenchymal carcinoma cells completely sensitizes otherwise refractory tumors to immunotherapy. While targeting cancer cell-intrinsic factors can sensitize mesenchymal tumors to ICB, the mechanisms that dictate partial versus complete responses are unclear. Furthermore, whether abrogation of CD73 can also sensitize mesenchymal tumors to other forms of ICB remains unknown. By performing immunofluorescent analysis of tumor sections, we observed that knockout of CD73 promoted the greatest infiltration of both CD4+ and CD8+ T-cells in response to anti-CTLA4 ICB compared to tumors knocked out for CSF1 and SPP1, which showed only intermediate levels of infiltration. Moreover, treatment of mesenchymal tumor-bearing mice with anti-CD73 generated synergistic responses with anti-CTLA4 ICB, but not with anti-PD1, or combinations of anti-CTLA4 and anti-PD1. In conclusion, our results demonstrate that the EMT program in cancer cells is predictive of responses to ICB. Targeting CD73 in mesenchymal breast tumors promotes an influx of both CD4+ and CD8+ T-cells and potentiates the efficacy of anti-CTLA4 ICB. Through mining of canine mammary tumor RNA sequencing data, we have found that canine tumors associated with EMT have increased expression of CD73, which suggests that these mechanisms of immune evasion may translate across species. These findings could promote therapies for both animals and humans that sensitize highly refractory populations of more-mesenchymal cancer cells. Citation Format: Kimaya M. Bakhle, Caitie H. Sams, Anushka Dongre. Targeting mesenchymal tumor cell-intrinsic factors sensitizes refractory tumors to immune checkpoint blockade therapy [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 6556.

Abstract 1750: Decoding breast cancer: Unraveling subtype and model differences through multi-model single-cell RNA sequencing data integration

Abstract Breast cancer's complex transcriptional landscape requires a deep understanding of sample and cell diversity to identify effective treatments. In this study, we amalgamate single-cell RNA sequencing data from breast cancer patient-derived xenografts (PDX), organoids, cell lines, patient tumors and reduction mammoplasties resulting in a comprehensive dataset of 117 samples with 506,719 total cells. These samples encompass hormone receptor positive (HR+), human epidermal growth factor receptor 2 enriched (HER2E), and triple-negative breast cancer (TNBC) subtypes. We aimed to delineate similarities and distinctions across model systems and patient samples while also exploring stratification of therapeutic drug efficacy based on subtype proportions within tumors. Mammary tumor PDXs, organoids, and established cell lines exhibited higher proliferation and lower heterogeneity observed via UMAP dimensionality reduction compared to most patient tumors or normal breast epithelium. TNBCs had elevated proliferative and pro-metastatic signatures compared to HR+ and HER2E samples. Interestingly, compared to matched PDX tumors, organoids from these same models were found to exhibit stark differences in gene expression, including upregulation of metabolically active aldo-keto reductase family genes, highlighting differences in the model systems with implications for pre-clinical drug testing. Single-cell tumor subtyping analyses with scSubtype and TNBCtype methods found that therapeutically treated samples had shifts in the proportions of cell-wise subtype annotations when compared to matched untreated samples. Similarly, patient lymph node metastasis when compared with matched primary tumors were significantly linked to decreases in Basal-like and HER2-enriched cell-wise annotations in untreated ER+ samples. In vitro assessment of anti-cancer compounds on PDX cells showed significant correlation of subtype proportion with cell viability following treatment with targeted therapeutic agents. This subtyping methodology offers a powerful tool to monitor the evolving gene expression landscape within samples and predict responses to therapeutic agents. We present here a dynamic approach to cell-wise sample annotation and a substantial multi-model dataset for use facilitating informed decision-making in preclinical research and therapeutic development. Citation Format: Julia E. Altman, Carson J. Walker, Emily K. Zboril, Nicole S. Hairr, Rachel K. Myrick, David C. Boyd, Jennifer E. Koblenski, Madhavi Puchalapalli, Bin Hu, Mikhail G. Dozmorov, Xi Chen, Yunshun Chen, Charles M. Perou, Brian D. Lehmann, Jane E. Visvader, Amy L. Olex, J. Chuck Harrell. Decoding breast cancer: Unraveling subtype and model differences through multi-model single-cell RNA sequencing data integration [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 1750.

Abstract 251: MCAM controls mammary cell state in luminal progenitor-derived breast cancer and alveologenesis

Abstract To establish and maintain functional and properly formed tissues, cells must integrate cues from their environment into fate decisions. External cues—from other cells and the extracellular matrix for instance—must be met with appropriate responses to ensure cell fate decisions that control lineage plasticity and favor proper development, maintenance, or regeneration of tissues while suppressing neoplastic transformation. MCAM (CD146; MUC18; S-endo-1; and Gicerin) is an integral membrane glycoprotein whose misexpression is associated with a variety of epithelial cancers, including breast cancer. However, the mechanisms by which MCAM conveys extracellular cues into malignant cellular outputs is not well understood. Our observation that MCAM is normally expressed in transplantable fetal and adult mammary stem cells (MaSCs) with multilineage potential, suggested it could control mammary cell state plasticity in both neoplastic and normal mammary tissue. Using a plastic, mouse mammary tumor cell line derived from the MMTV-PyMT mouse (Py230) that has been reported to exhibit mammary stem cell and luminal progenitor features, we tested the role of Mcam in controlling the tumorigenicity and lineage/cell-state relationships of mammary tumor cells. We found that Mcam knockdown in Py230 cells increases STAT3 expression, activation, and downstream target transcription, leading to a shift in the predominant cell state away from a luminal progenitor state toward more differentiated alveolar and basal phenotypes. Mechanistically, these changes involve altered activity of casein kinase II (CK2) against multiple substrates including STAT3 and PTEN. This effect may be mediated by direct coupling and sequestration of CK2 to various substrates via the cytoplasmic tail of MCAM. We have also found that inhibition of Ck2 or Stat3 reversed the transcriptional state change elicited by Mcam knockdown. In grafted tumors in vivo, this state switch correlates with reduced overall tumorigenicity and attenuated expression of Sox10, a gatekeeper of neural crest-like invasive features. In the normal gland, we hypothesize that this activity regulates multilineage stem cell potential upon transplantation and multilineage regenerative potential across lactation/involution cycles as we found reciprocal expression patterns for MCAM and STAT3 in alveolar mammary epithelial cells. These data point to an integral role for MCAM in lineage plasticity of normal mammary epithelial cells that is co-opted during transformation to potentiate tumor cell plasticity, progression, and evasion of targeted cancer therapies. Citation Format: Brooke L. Gates, Ozlen Balcioglu, David W. Freeman, Berhane M. Hagos, Benjamin T. Spike. MCAM controls mammary cell state in luminal progenitor-derived breast cancer and alveologenesis [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 251.

Abstract 76: Correlated multi-scale 3D optical microscopy of tumor tissues

Abstract Three-dimensional (3D) optical microscopy in combination with advanced tissue clearing methods permits the interrogation of intact large-sized tumor tissues. There are many optical microscopy methods (e.g. light-sheet and confocal microscopy) offering 3D tumor images at varying levels of spatial resolution (e.g. organ, tissue, cell, and molecule level). However, it is a challenge to correlatively integrate various resolution 3D images obtained by different optical microscopy of tumor tissue. In this project, we developed a correlated multi-scale 3D optical microscopy method that enables the acquisition of macroscopic to microscopic spatial information from a single tumor tissue in 3D. Firstly, we used light sheet microscopy to image a whole large-sized, cleared tumor tissue and analyzed the results to identify regions of interests (ROIs). After defining ROIs in the 3D whole tumor image, we added a UV-activatable visible dye, spiropyran, in the agarose gel surrounding the cleared tumor and exposed the sample to a UV light sheet horizontally at the height (z-axis) of the tumor where the ROIs present. The activated dye showed ‘purple’ colored lines in the agarose gel and we physically marked the gel along the visible colored lines. We further sectioned the tumor at the ROI positions using a vibratome after reversing tissue clearing process. After immunofluorescence staining and aqueous-based tissue clearing, confocal microscopy allowed the localization of various cell types in the ROIs of tumor macrosections. To validate the method for multi-scale evaluation of cancer immunotherapy, we tested it with control and DMXAA-treated mouse mammary tumor tissues. We successfully acquired the 3D images of whole tumors by capturing autofluorescence signals with a 633 nm excitation laser and its matched emission filter. Based on the autofluorescence tumor images, we defined two ROIs for each control and treated tumor and performed optical marking and physical sectioning of the tumors as described above. Next, we stained the macrosections (400 µm thick) with DAPI, CK8-DL488, CD45-DL550, and ER-TR7-DL680 to visualize cancer, immune, and stromal cells in the ROIs at cellular resolution. We quantitatively analyzed the correlated multi-scale 3D image data for assessment of the effects of DMXAA treatment on the tumor microenvironment including eradiation of cancer cells, immune infiltration, and remodeling of tumor stroma. In summary, this new microscopy method will provide comprehensive spatial information of tumor tissues and benefit a broad cancer research field such as the discovery of new tumor biomarkers and the development of effective cancer therapies. Citation Format: Jingtian Zheng, Steve Seung-Young Lee. Correlated multi-scale 3D optical microscopy of tumor tissues [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 76.

Abstract 5910: Developing a novel humanized anti-FABP4 antibody for breast cancer treatment

Abstract Circulating levels of adipocyte fatty acid binding protein (A-FABP, also known as FABP4) are linked with metabolic dysregulation. Our previous study demonstrated that high serum levels of FABP4 increased the risk of breast cancer, indicating that circulating FABP4 could be a potential therapeutic target for this disease. In this study, we generated mouse monoclonal antibodies (mAb) against FABP4 by immunizing C57BL/6 mice with recombinant human FABP4. After screening over 1300 hybridoma clones, we identified an FABP4 neutralizing mAb, named 12G2, which inhibited breast cancer growth in various mouse models. To facilitate clinical application, we engineered chimeric and humanized variants of the 12G2 mAb. Notably, the humanized variant, 12G2-variant 9 (12G2-vt9), effectively inhibited mammary tumor growth and progression by reducing the activity of FABP4/ALDH1 in different breast cancer mouse models. Our findings suggest that 12G2-Vt9 is a promising neutralizing mAb targeting FABP4-mediated cancer stemness through ALDH1 signaling, offering potential as a targeted therapeutic antibody for the clinical treatment of breast cancer. Citation Format: Jiaqing Hao, Matthew Yorek, Jian yu, Anthony Avellino, Bing Li. Developing a novel humanized anti-FABP4 antibody for breast cancer treatment [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 5910.

Abstract 1127: Single cell network-based analyses reveal dose-dependent increase in myeloid suppressive cell sub-populations in response to radiation therapy

Abstract Background: More than half of all cancer patients undergo radiation therapy (RT) with curative or palliative intent. With the widespread use of immunotherapy to treat cancer, immune effects of radiation are an important consideration in design of sequential and combination therapies. While radiation has been found to deplete lymphocytes, less is known about its effects on the myeloid compartment, which includes both immune activating and suppressive populations. We hypothesized that a network-based analysis of single cell proteogenomic data generated from tumor-infiltrating immune cells can identify druggable proteins in pathways driving unwanted immunosuppressive changes with radiation, particularly among myeloid cells. Methods: Proteogenomic (CITE-Seq) analysis was conducted on CD45+ immune cells from unirradiated and irradiated orthotopic 4T1 murine mammary tumors three- and ten-days after tumor irradiation (8 Gy x 1 or 8 Gy x 3). Based on expression of select surface proteins, analysis of the CITE-Seq data resulted in eight distinct immune clusters, including three myeloid clusters — monocytes, macrophages, and granulocytes. Using VIPER (virtual inference of protein activity by enriched regulon), an algorithm that infers protein activities from weighted expression on each protein’s downstream targets, we identified distinct functional subpopulations within these eight clusters. Further profiling subclusters enriched by RT using the OncoTarget algorithm, we identified druggable proteins with significant inferred activity as potential targets of therapeutic interest. Results: As expected, lymphocytes were persistently depleted after RT on both radiotherapy schedules. However, three distinct subclusters of myeloid cells with immune-suppressive phenotype became enriched following tumor irradiation, with greater enrichment at the lower radiation dose of 8 Gy x 1: TAM2, PMN1, and PMN4. Surface protein expression enabled us to define the immunophenotype and isolate these three cell clusters from murine 4T1 tumors as well as human renal cell carcinoma tumors for further functional analyses. OncoTarget identified a number of druggable targets, including PARP1 for the PMN1 subcluster. Conclusion: Suppressive myeloid subpopulations induced by radiation were identified among the immune cells. Combining radiation therapy with inhibitors of these cell populations may provide therapeutic benefit over radiation therapy alone or in combination with checkpoint inhibitor immunotherapies. Future work will experimentally determine if inhibition of druggable protein targets in these myeloid subpopulations improves the overall efficacy of RT. Citation Format: Aparna Krishnan, Shruti Bansal, Samanta Sarti, Michael D. Kissner, Charles Karan, Andrea Califano, Aleksandar Z. Obradovic, Catherine S. Spina. Single cell network-based analyses reveal dose-dependent increase in myeloid suppressive cell sub-populations in response to radiation therapy [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 1127.

Abstract 261: Akt1 and Cyclin D1 govern polarity of self-renewing divisions in breast cancer stem cells

Abstract Background. Stem-like cells contribute to the development and progression of human cancers. Genes that control symmetric cell divisions have an evolutionarily conserved role in the regulation of cell polarity and in tumorigenesis. Two cells generated by asymmetric divisions have reduced proliferative potential compared with symmetric divisions. The cell survival oncoprotein Akt (Protein Kinase B) is encoded by three isoforms that are altered in human cancers. The Akt isoform-specific impact on breast cancer stem cell function and polarity is controversial, with Akt1 either reducing (1) or promoting (2) cell polarity and migration in tissue culture. Deletion of Akt1 reduced mammary tumor progression and metastasis in transgenic mice (2,3). Methods. Analysis of Akt1, cyclin D1 and Notch signaling was conducted from public databases, and human as well as murine breast cancer samples. Cells derived from Akt1 gene deletion MMTV-ErbB2 breast oncomice (MMTV-ErbB2/Akt1+/+ and MMTV-ErbB2/Akt1−/−) and from the human MCF-7 breast cancer cell line with regulated expression of cyclin D1 and p53 were analyzed for the symmetry of cell division, stem cell function, and signaling pathways. Results. In breast tumors derived from MMTV-ErbB2 transgenics, associated with reduced metastasis, Akt1 genetic deletion reduced the frequency of symmetric cell divisions and the production of mammospheres. Akt1 deletion increased p53 and reduced cyclin D1. Pharmacological reactivation of p53 with Nutlin correlated with the reinduction of asymmetric cellular divisions and reduction in mammospheres in murine and human BCa cells. Akt1 deletion abrogated Nutlin-mediated induction of asymmetric cell divisions. Cyclin D1 induced symmetrical cell divisions, mammosphere formation and reduced p53 and Numb. Conclusions. Akt1 promoted symmetrical cell division at least in part via cyclin D1 suppression of Numb, and induction of γ secretase activity and thereby Notch signaling. Reference. (1) Irie HY, Pearline RV, Grueneberg D, Hsia M, Ravichandran P, Kothari N, et al. Distinct roles of Akt1 and Akt2 in regulating cell migration and epithelial-mesenchymal transition. J Cell Biol 2005;171:1023-34. (2) Ju X, Katiyar S, Wang C, Liu M, Jiao X, Li S, et al. Akt1 governs breast cancer progression in vivo. Proc Natl Acad Sci U S A 2007;104:7438-43. (3) Chen X, Ariss MM, Ramakrishnan G, Nogueira V, Blaha C, Putzbach W, et al. Cell-Autonomous versus Systemic Akt Isoform Deletions Uncovered New Roles for Akt1 and Akt2 in Breast Cancer. Mol Cell 2020;80:87-101 e5 Citation Format: Xuanmao Jiao, Xin Sun, Zhiping Li, Xiaoming Ju, Danni Li, Ritika Harish, Gordon Robertson, Anthony W. Ashton, Richard G. Pestell. Akt1 and Cyclin D1 govern polarity of self-renewing divisions in breast cancer stem cells [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 261.

Abstract 5364: Progesterone receptor modulates the antigen processing and presentation machinery decreasing MHC class I expression on tumors

Abstract Hormone receptor (HR) positive breast cancers, which account for approximately 75% of all breast cancers and express both estrogen receptor (ER) and progesterone receptor (PR), have been the subject of much research. As such, successful anti-estrogen/ER-based therapies have been very successful in the treatment of HR+ breast cancer. Despite this success, over 1/3 of patients will eventually progress on current ER/estrogen-targeted therapies, underscoring the need for new targeted therapies in HR+ breast cancer. Unlike ER, the role of PR in breast cancer progression has received far less attention. In a previous study, we found that a mouse mammary tumor cell line E0771, modified to express the mouse progesterone receptor (mPR) and the OVA peptide, demonstrated decreased T cell-mediated cell death when treated with progesterone compared to the control group. Progesterone-mediated protection from T-cell death was only observed in mPR+ cell lines. Flow analysis of the E0771-Ova-mPR cells also revealed a decrease in MHC Class I presentation in progesterone-treated cells compared to a vehicle control. To further explore the role of the machinery required for antigen processing and presentation of MHC Class I molecules, we conducted additional experiments using the E0771 cell line. Our results showed that treatment with progesterone led to a reduction in RNA levels of all the machinery required for antigen processing and presentation (APP). Based on these findings, we hypothesize that activation of the progesterone receptor by progesterone may decrease MHC Class I expression by regulating APP. Our results showed that treatment with progesterone resulted in a reduction in RNA levels of Tap1, Tap2, Tapbp, Nlrc5, B2m, and Psmb8, which are essential for APP. We therefore propose that activation of the progesterone receptor by progesterone may decrease MHC Class I expression by directly modulating the APP pathway. The findings of this study may have significant implications for our understanding of how breast cancer cells evade T-cell mediated cytotoxicity. Citation Format: Julio Tinoco, Lauryn Werner, Eilidh Chowanec, Harmony Saunders, Sun Xiaopeng, Jay Gertz, Justin Balko, Zachary C. Hartman, Christy Hagan. Progesterone receptor modulates the antigen processing and presentation machinery decreasing MHC class I expression on tumors [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 5364.

Abstract 1366: Dual immune checkpoint inhibition enhances the anti-tumor activity of trastuzumab deruxtecan in preclinical models

Abstract Background Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate (ADC) composed of an anti-human epidermal growth factor receptor 2 (HER2) antibody, a cleavable tetrapeptide-based linker, and a topoisomerase I inhibitor payload. Recent clinical trials such as BEGONIA have highlighted the potential therapeutic benefit in combining T-DXd with immuno-oncology (IO) agents such as the PD-L1 inhibitor durvalumab. Here, we report preclinical findings demonstrating enhanced anti-tumor activity when T-DXd is combined with dual immune checkpoint inhibition. Methods HER2-expressing human cancer cell lines were treated in vitro with T-DXd and assessed for induction of immunogenic cell death (ICD) markers and expression of NK cell-activating ligands. Human macrophages and T cells were incubated with supernatants from treated cancer cells, and their activation status evaluated via flow cytometry. In vivo, the effects of T-DXd in combination with inhibitors of PD-L1, CTLA-4, and a monovalent bispecific anti-PD-1/TIGIT antibody - a murine surrogate of rilvegostomig - on tumor growth were evaluated in BALB/c mice bearing human HER2-low EMT6 murine mammary tumors. The anti-tumor activity of T-DXd in combination with volrustomig, a monovalent bispecific antibody targeting PD-1 and CTLA-4, was also assessed in hu-CD34+ NSG mice bearing HER2-low Caki-1 renal cell carcinoma tumors. Results In vitro, T-DXd induced ICD via DXd-mediated extracellular release of inflammatory mediators ATP and HMGB1, and cell surface exposure of calreticulin. This was associated with a greater than 2-fold increase in expression of NK cell-activating ligands, and activation of both macrophages and T cells. In immunocompetent mice bearing human HER2 expressing EMT6 tumors, T-DXd treatment drove tumor growth inhibition (53%, P <0.001) that was associated with an increase in tumoral T cells (2.6-fold, P <0.05) and increased CD8+ T cell expression of the immune checkpoints PD-1, TIGIT, and TIM-3. Tumor growth inhibition relative to vehicle-treated mice was enhanced when T-DXd was combined with inhibitors of PD-L1 (108%, P <0.001), PD-L1 plus CTLA-4 (172%, P <0.001), or a bispecific PD-1/TIGIT inhibitor (181%, P <0.001). Pharmacodynamic analysis revealed how the combination of T-DXd and PD-1/TIGIT inhibition increased tumoral NK cells (3.2-fold, P <0.001) and CD8+ T cells (3.8-fold, P <0.001) relative to vehicle. Consistent with findings in syngeneic models, the combination of T-DXd with a bispecific PD-1/CTLA-4 inhibitor also enhanced tumor growth inhibition in a humanised Caki-1 model (121%) compared to T-DXd alone (72%) (P <0.001). Conclusions These data provide insight into the clinical activity observed with T-DXd and provide scientific rationale for combination strategies with novel, bispecific IO agents targeting CTLA-4 or TIGIT in addition to the PD-1/PD-L1 axis. Citation Format: Liam Jenkins, Laura Kazlauskas, Matt Wilson, Scott A. Hammond, Theresa Proia, Jerome Mettetal. Dual immune checkpoint inhibition enhances the anti-tumor activity of trastuzumab deruxtecan in preclinical 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 1366.

Abstract 293: Distinctive roles of mammary adipomes in breast cancer progression: Insights from functional analysis and differential gene expression

Abstract Breast Cancer (BC) is a heterogeneous disease that impacts treatment response. This is due in part to the tumor microenvironment (TME), which contributes significantly to BC pathogenesis, including metastasis. Women with higher breast density and reduced breast fat face an increased BC risk across multiple tumor subtypes. This is exemplified by the correlation between increased tumor progression, the appearance of fibrosis and the loss of mature adipocytes. While the role of adipokines in this process has been studied extensively, the contribution of adipocyte-derived extracellular vesicles (EV) or "adipomes" in tumorigenesis and metastasis remains poorly understood. To address this question, we developed a novel technique for isolating intact adipomes from normal mammary fat (MF) obtained from wild-type C57BL/6 mice as well as from tumor-associated MF (TAMF) obtained from mice inoculated orthotopically with EO771 mammary tumor cells. Adipome isolation involved high-speed centrifugation to separate macro- and micro-vesicles, purification using the ExoQuick EV isolation system, and immunomagnetic capture of adipomes. Characterization of adipomes by transmission electron microscopy revealed two adipome populations, large L-adipomes of ~350 nm, and small S-adipomes of ~40 nm. Notably, the quantity of L-adipomes in tumor-associated mammary fat (TAMF) was 10-fold greater than the amount of L-adipomes in the MF of non-tumor-bearing mice. To identify possible functional roles of L-adipomes in the pathogenesis of BC, lipidomic profiling was undertaken. L-Adipomes from TAMF showed significantly altered lipid species in comparison to MF-derived L-adipomes. Adipomes were further characterized by treating EO771 cells for 72 hr with TAMF- or MF-derived adipomes at a cell to adipome ratio of 1:10, followed by RNAseq analysis. Differentially upregulated genes in TAMF-derived L-adipomes included Esr1, Erbb2, Tgfb1, Bcl2, MMP2, and Notch associated with fibrosis, EMT, angiogenesis and invasion and downregulation of genes linked to tumor suppression, apoptosis, and mitochondrial metabolism. Overall, our study illustrates the importance of tumor-associated adipomes in regulating gene expression in tumor cells, which emphasizes the specificity of adipome content relating to the cell-of-origin and the host's pathologic and metabolic state and in modulating signaling pathways involved in BC progression. Citation Format: Hariprasad Thangavel, Kezia Lizardo, Robert I. Glazer, Jyothi F. Nagajyothi. Distinctive roles of mammary adipomes in breast cancer progression: Insights from functional analysis and differential gene expression [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 293.

Abstract 6628: Mapping the spatial and molecular determinants of macrophage heterogeneity in breast cancer

Abstract Tumor-infiltrating macrophages support critical steps in tumor progression, and their accumulation in the tumor microenvironment (TME) is associated with adverse outcomes and therapeutic resistance across human cancers. In the TME, macrophages adopt diverse phenotypic alterations, giving rise to heterogeneous immune activation states and induction of cell cycle. While the transcriptional profiles of these activation states are well-annotated across human cancers, the underlying signals that regulate macrophage heterogeneity and accumulation remain incompletely understood. Here, we leveraged a novel ex vivo organotypic TME (oTME) model of breast cancer, in vivo murine models, and human samples to map the determinants of functional heterogeneity of TME macrophages. We identified a subset of F4/80high Sca-1+ self-renewing macrophages maintained by type-I interferon (IFN) signaling and requiring physical contact with cancer-associated fibroblasts. We discovered that the contact-dependent self-renewal of TME macrophages is mediated via Notch4, and its inhibition abrogated tumor growth of breast and ovarian carcinomas in vivo, as well as lung dissemination in a PDX model of triple-negative breast cancer (TNBC). Through spatial multi-omic profiling of protein markers and transcriptomes, we found that the localization of macrophages further dictates functionally distinct but reversible phenotypes, regardless of their ontogeny. Whereas immune-stimulatory macrophages (CD11C+CD86+) populated the tumor epithelial nests, the stroma-associated macrophages (SAMs) were proliferative, immunosuppressive (Sca-1+CD206+PD-L1+), resistant to CSF-1R depletion, and associated with worse patient outcomes. Notably, following cessation of CSF-1R depletion, macrophages rebounded primarily to the SAM phenotype, which was associated with accelerated growth of mammary tumors. Our work reveals the spatial determinants of macrophage heterogeneity in breast cancer and highlights the disruption of macrophage self-renewal as a potential new therapeutic strategy. Citation Format: Nir Ben Chetrit, Xiang Niu, Jesus Sotelo, Ariel Swett, Vinagolu Rajasekhar, Maria Jiao, Caitlin Stewart, Priya Bhardwaj, Sanjay Kottapalli, Saravanan Ganesan, Pierre-Louis Loyher, Catherine Potenski, Kristy Brown, Neil M. Iyengar, Dilip D. Giri, Scott W. Lowe, John H. Healey, Frederic Geissmann, Irit Sagi, Johanna A. Joyce, Dan A. Landau. Mapping the spatial and molecular determinants of macrophage heterogeneity 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 6628.

Abstract 1499: Senescent stromal cells drive lung metastases of breast cancer

Abstract In 2022 in the United States, there were nearly 300,000 women diagnosed with breast cancer (BC) with the risk increasing significantly after age 50. The overall 5-year survival rate dramatically decreases, from 99% for patients with stage I, to a devastating 25% for those with stage IV (metastatic) BC, making distal metastasis the leading cause of BC mortality. This mortality rate has not changed in decades with lungs being one of the most common sites for distal metastasis of BC. The BC microenvironment is a complex mix, consisting of tumor cells and numerous nontumor cells including distinct subpopulations of cancer-associated fibroblasts (CAFs): vascular (vCAFs), inflammatory (iCAFs), and myofibroblastic CAFs (myCAFs), which can play pro- or anti-tumor roles. Senescent fibroblasts, which accumulate with age typically express p16INK4A and can modulate the cells around them via the secretion of a cadre of pro-tumorigenic factors including cytokines, chemokines, and extracellular matrix (ECM) modulating enzymes, collectively called the senescence-associated secretory phenotype (SASP). To investigate how p16+ senescent cells impact tumorigenesis, we mated the well-characterized MMTV-PyMT mouse, to the INK-ATTAC (INK) mouse, wherein treatment with AP20187 (AP) selectively eliminates p16+ senescent cells. Preliminary data showed that elimination of p16+ aSMA+ senescent fibroblasts (senCAF) resulted in a significant decrease in primary tumors, lung tumor foci and lung metastatic burden. Further, we found that senescent CAFs (senCAFs) were the only stromal cells eliminated in the PyMT-INK mouse treated with AP, suggesting that this CAF subtype is a key contributor to tumor progression. To determine if senCAFs directly impacted lung metastasis, EO771-LG Luc mammary tumor cells were injected in the tail vein of INKATTAC mice that were treated with AP to eliminate p16+ senCAFs. We found that metastatic tumor burdens were significantly reduced in INK+ versus control mice and immunohistochemical analysis revealed that tumor burden and foci was significantly reduced, suggesting that senCAFs in the lung promoted tumor cell seeding and outgrowth. Citation Format: Anupama Melam, Jiayu Ye, Sheila A. Stewart. Senescent stromal cells drive lung metastases of 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 1499.

Engineered Hybrid Vesicles and Cellular Internalization in Mammary Cancer Cells.

Extracellular vesicles play an important role in intercellular communication, with the potential to serve as biomaterials for nanocarriers. Combining such extracellular vesicles and liposomes results in advanced drug delivery carriers. In this study, we attempted to fabricate hybrid vesicles using a membrane fusion method and incorporated an anticancer drug. As a result, we successfully prepared nanosized uniform hybrid vesicles and evaluated their physicochemical characteristics and intracellular uptake mechanisms via endocytosis in various cell lines. Compared to liposomes, the hybrid vesicles showed better physical properties and a relatively higher reduction in cell viability, which was presumably dependent on the specific cell type. These findings suggest that fusion-based hybrid vesicles offer a novel strategy for delivering therapeutic agents and provide insights into the types of extracellular vesicles that are useful in fabricating hybrid vesicles to develop an advanced drug delivery system.

Abstract 7460: Characterizing the role of PCBP1 in the activation of the cGAS-STING/type I Interferon axis to promote anti-tumor immunity

Abstract The emergence of immunotherapies has encouraged the need to better understand the molecular mechanisms that modulate the tumor immune microenvironment to kill cancer cells as well as the strategies used by cancer cells to evade the immune system. In several cancer types, the Poly(rC) Binding Protein 1 (PCBP1) has shown to have tumor suppressive effects, however its function in regulating the tumor immune environment remains unclear. To investigate this in the context of breast cancer, we established a CRISPR-Cas9-medited knock-out of PCBP1 in a Py8119 murine breast cancer cell line. PCBP1−/− cells revealed a decrease in the activation of the cGAS-STING pathway and is associated with lower levels of type I interferons (IFNs) and Interferon Stimulated Genes’ (ISGs) expression. The cGAS-STING pathway plays an important role in innate immunity as it fights pathogenic infections and tumorigenesis through the production of type I IFN and ISGs. It also promotes the priming and infiltration of T cells at the tumor site to fight cancer cells. Our results suggested that PCBP1 is acting on the activation of the cGAS-STING pathway and could further modulate the tumor immune microenvironment. We then confirmed our results in vivo by establishing a tumorigenic C57BL/6 PyMT transgenic mouse model with conditional knockout for PCBP1 in mammary cells. Our data demonstrate that PCBP1−/− PyMT mice have higher tumor burdens compared to PCBP1+/+ PyMT mice and also show a decreased ISGs’ signature by RNA-sequencing, qPCR and western blot, as previously observed in vitro in the Py8119 PCBP1−/− cell line. Additional single nuclei RNA-sequencing analyses confirmed that this downregulation of ISGs is observed in mammary cancer cells where PCBP1 is knocked-out. Strikingly, IHC and flow cytometry analyses of PCBP1−/− tumors isolated from these mice also revealed a decrease in CD8+ T-cells. Therefore, PCBP1 silencing may promote tumor growth by impairing the activation of the cGAS-STING/IFN-I/ISGs axis, which may contribute to the decrease in CD8+ T-cells’ infiltration at the tumor site. Additionally, we are further investigating the molecular mechanisms mediated by PCBP1 that trigger this activation of the cGAS-STING pathway. Citation Format: Cécile Fréreux, Paramita Chakraborty, Annamarie C. Dalton, Breege V. Howley, Bryan Granger, Joseph A. Karam, Silvia G. Vaena, Martin J. Romeo, Philip H. Howe. Characterizing the role of PCBP1 in the activation of the cGAS-STING/type I Interferon axis to promote anti-tumor immunity [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 7460.

Convergence between helminths and breast cancer: intratumoral injection of the excretory/secretory antigens of the human parasite Toxocara canis (EST) increase lung macro and micro metastasis.

Worldwide, breast cancer is the most important cancer in incidence and prevalence in women. Different risk factors interact to increase the probability of developing it. Biological agents such as helminth parasites, particularly their excretory/secretory antigens, may play a significant role in tumor development. Helminths and their antigens have been recognized as inducers or promoters of cancer due to their ability to regulate the host's immune response. Previously in our laboratory, we demonstrated that chronic infection by Toxocara canis increases the size of mammary tumors, affecting the systemic response to the parasite. However, the parasite does not invade the tumor, and we decided to study if the excretion/secretion of antigens from Toxocara canis (EST) can affect the progression of mammary tumors or the pathophysiology of cancer which is metastasis. Thus, this study aimed to determine whether excretion/secretion T. canis antigens, injected directly into the tumor, affect tumor growth and metastasis. We evaluated these parameters through the monitoring of the intra-tumoral immune response. Mice injected intratumorally with EST did not show changes in the size and weight of the tumors; although the tumors showed an increased microvasculature, they did develop increased micro and macro-metastasis in the lung. The analysis of the immune tumor microenvironment revealed that EST antigens did not modulate the proportion of immune cells in the tumor, spleen, or peripheral lymph nodes. Macroscopic and microscopic analyses of the lungs showed increased metastasis in the EST-treated animals compared to controls, accompanied by an increase in VEGF systemic levels. Thus, these findings showed that intra-tumoral injection of T. canis EST antigens promote lung metastasis through modulation of the tumor immune microenvironment.