Syngeneic Mice Research Articles

Discovery of the Selective and Orally Available Galectin-1 Inhibitor GB1908 as a Potential Treatment for Lung Cancer.

We have previously described a new series of selective and orally available galectin-1 inhibitors resulting in the thiazole-containing glycomimetic GB1490. Here, we show that the introduction of polar substituents to the thiazole ring results in galectin-1-specific compounds with low nM affinities. X-ray structural analysis of a new ligand-galectin-1 complex shows changes in the binding mode and ligand-protein hydrogen bond interactions compared to the GB1490-galectin-1 complex. These new high affinity ligands were further optimized with respect to affinity and ADME properties resulting in the galectin-1-selective GB1908 (Kd galectin-1/3 0.057/6.0 μM). In vitro GB1908 inhibited galectin-1-induced apoptosis in Jurkat cells (IC50 = 850 nM). Pharmacokinetic experiments in mice revealed that a dose of 30 mg/kg b.i.d. results in free levels of GB1908 in plasma over galectin-1 Kd for 24 h. GB1908 dosed with this regimen reduced the growth of primary lung tumor LL/2 in a syngeneic mouse model.

Inhibition of P21-activated kinases 1 and 4 synergistically suppresses the growth of pancreatic cancer by stimulating anti-tumour immunity

BackgroundPancreatic ductal adenocarcinoma (PDA) is one of the most lethal types of cancer, and KRAS oncogene occurs in over 90% of cases. P21-activated kinases (PAK), containing six members (PAK1 to 6), function downstream of KRAS. PAK1 and PAK4 play important roles in carcinogenesis, but their combinational effect remains unknown. In this study, we have determined the effect of dual inhibition of PAK1 and PAK4 in PDA progression using knockout (KO) cancer cell lines.MethodsMurine wild-type (WT) and PAK1KO pancreatic cancer cell lines were isolated from PAK1+/+ and PAK1−/− KPC (LSL-KrasG12D/+; LSL-Trp53 R172H/+; Pdx-1-Cre) mice. KPC PAK4KO and KPC PAK1&4 KO cell lines were generated from KPC WT and KPC PAK1KO cell lines respectively using the CRISPR-CAS9 gene knockout technique. PAK WT and KO cell lines were used in mouse models of pancreatic tumours. Cells and tumour tissue were also used in flow cytometry and proteomic studies. A human PDA tissue microarray was stained by immunohistochemistry.ResultsDouble knock out of PAK1 and PAK4 caused complete regression of tumour in a syngeneic mouse model. PAK4KO inhibited tumour growth by stimulating a rapid increase of cytotoxic CD8+ T cell infiltration. PAK1KO synergistically with PAK4KO increased cytotoxic CD8+ T cell infiltration and stimulated a sustained infiltration of CD8+ T cells at a later phase to overcome the immune evasion in the PAK4KO tumour. The human PDA tissue microarray study showed the important role of PAK1 and PAK4 in intra-tumoral T-cell function.ConclusionOur results demonstrated that dual inhibition of PAK1 and PAK4 synergistically suppressed PDA progression by stimulating cytotoxic CD8 + T cell response.

Mitotic MTH1 inhibitor TH1579 induces PD-L1 expression and inflammatory response through the cGAS-STING pathway

The mitotic MTH1 inhibitor TH1579 is a dual inhibitor that inhibits mitosis and incorporation of oxidative DNA damage and leads to cancer-specific cell death. The response to immune checkpoint inhibitor (ICI) treatment is often augmented by DNA damaging agents through the cGAS-STING pathway. This study investigates whether TH1579 can improve the efficacy of immune checkpoint blockades through its immunomodulatory properties. Various human and murine cancer cell lines were treated with mitotic MTH1i TH1579, and the expression of PD-L1 and T-cell infiltration-related chemokines was analysed by flow cytometry and real-time qPCR. Syngeneic mouse models were established to examine the combined effect of TH1579 and PD-L1 blockade. In our investigation, we found that TH1579 upregulates PD-L1 expression at both the protein and mRNA levels in human cancer cell lines. However, in murine cell lines, the increase was less pronounced. An in vivo experiment in a syngeneic mouse melanoma model showed that TH1579 treatment significantly increased the efficacy of atezolizumab, an anti-PD-L1 antibody, compared to vehicle or atezolizumab monotherapy. Furthermore, TH1579 exhibited immune-modulatory properties, elevating cytokines such as IFN-β and chemokines including CCL5 and CXCL10, in a cGAS-STING pathway-dependent manner. In conclusion, TH1579 has the potential to improve ICI treatment by modulating immune checkpoint-related proteins and pathways.

Synergistic effects of Smac mimetic APG-1387 with anti-PD-1 antibody are attributed to increased CD3 + NK1.1 + cell recruitment secondary to induction of cytokines from tumor cells

BackgroundImmune checkpoint inhibitors are approved for the treatment of various tumors, but the response rate is not satisfactory in certain malignancies. Inhibitor of apoptosis proteins (IAP) ubiquitin-E3 ligase activity is involved in the regulation of immune responses. APG-1387 is a novel second mitochondria-derived activator of caspase (Smac) mimetic IAP inhibitor. The aim of this study was to explore the synergistic effect of APG-1387 when combined with anti-PD-1 antibody in a preclinical setting.MethodsWe utilized syngeneic mouse models of ovarian cancer (ID8), colon cancer (MC38), malignant melanoma (B16), and liver cancer (Hepa1-6) to assess the combination effect of APG-1387 and anti-PD-1 antibody, including immune-related factors, tumor growth, and survival. MSD V-PLEX validated assays were used to measure in vitro and in vivo cytokine release.ResultsIn ID8 ovarian cancer and MC38 colon cancer models, APG-1387 and anti-PD1 antibody had synergistic antitumor effects. In the MC38 model, the combination of APG-1387 and anti-PD-1 antibody significantly inhibited tumor growth (P < 0.0001) and increased the survival rate of tumor-bearing animals (P < 0.001). Moreover, we found that APG-1387 upregulated tumor-infiltrating CD3 + NK1.1 + cells by nearly 2-fold, by promoting tumor cell secretion of IL-12. Blocking IL-12 secretion abrogated the synergistic effects of APG-1387 and anti-PD-1 antibody in both MC38 and ID8 models.ConclusionsAPG-1387 has the potential to turn “cold tumors” into hot ones by recruiting more CD3 + NK1.1 + cells into certain tumors. Based on these and other data, the safety and therapeutic effect of this combination will be investigated in a phase 1/2 trial in patients with advanced solid tumors or hematologic malignancies (NCT03386526).Graphic abstract

Targeted Clearance of Senescent Cells via Engineered Extracellular Vesicles Reprograms Tumor Immunosuppressive Microenvironment.

Unravelling the mechanisms for the immunosuppressive tumor microenvironment and developing corresponding therapeutic strategies are of great importance to improve the cancer immunotherapy. In this study, we have revealed that there are abundant senescent cells accumulated in the colon cancer tissue, which contributes greatly to the immunosuppressive microenvironment. Oral delivery of Dasatinib and Quercetin (D+Q) eliminates the senescent cells with compromised efficiency due to the poor tumor penetration and short half-life. To improve the efficacy of senescent cell clearance, we have developed an extracellular vesicle (EV)-based senolytic strategy. The engineered senolytic EVs have anti-GPNMB (a senescent cell surface marker) displayed on the surface and D+Q loaded on the membrane. In a syngeneic mouse model, senolytic EVs efficiently and selectively eradicate the senescent cells and in turn unleashes the antitumor immunity. With the antitumor immunity boosted, cancer growth is inhibited and the survival is prolonged. In summary, we here have illuminated that senescent cells contribute to the immunosuppressive microenvironment in colon cancer and proposed a novel strategy to conquer the problem by EV-based senolytics. This article is protected by copyright. All rights reserved.

Cell based and In vivo systematic evaluation of some Egyptian plant extracts targeting breast cancer

The prevalence of breast cancer as a significant public health concern necessitates continued exploration of natural resources for novel anti-cancer agents is crucial. Material and methodsAnticancer activity of plant extracts on monolayer breast cancer cell line (MCF7) with lower levels of toxicity towards normal (RPE1) underwent further assessment using a three-dimensional model (3D). The extract's effects were investigated through multiple assays including apoptosis induction using quantifying cleaved cytokeratin-18 (CK18) and DNA fragmentation. Additionally, the expression of Bcl-2 and Bax was quantitative using real-time PCR. The median lethal dose (LD50) was determined by the acute oral toxicity, while biomarkers associated with tumorigenesis, metastasis, and cell death were quantified by ELISA. ResultsLimoniastrum monopetalum and Bauhinia variegata exhibited the most potent antitumor efficacy among the investigated extracts. They demonstrated potent cytotoxicity against MCF7 with no significant effect on hTERT RPE-1, with an IC50 of 100 μM. The extract demonstrated effectiveness in killing cancer cells within 3D tumor-like structures, induced apoptosis through caspase-3 activation and cleavage of cytokeratin-18, up-regulated the tumor suppressor p53, down-regulated the anti-apoptotic Bcl-2 gene, and caused DNA fragmentation. Acute oral toxicity studies in mice indicated low toxicity, and in a syngeneic mouse tumor model, the extract significantly inhibited tumor growth, suggesting its potential for further development. ConclusionLimoniastrum monopetalum and Bauhinia variegata exhibited the most potent antitumor efficacy among the investigated extracts.

Synergistic antitumor activity by dual blockade of CCR1 and CXCR2 expressed on myeloid cells within the tumor microenvironment.

Chemokine signaling within the tumor microenvironment can promote tumor progression. Although CCR1 and CXCR2 on myeloid cells could be involved in tumor progression, it remains elusive what effect would be observed if both of those are blocked. We employed two syngeneic colorectal cancer mouse models: a transplanted tumor model and a liver metastasis model. We generated double-knockout mice for CCR1 and CXCR2, and performed bone marrow (BM) transfer experiments in which sub-lethally irradiated wild-type mice were reconstituted with BM from either wild-type, Ccr1-/-, Cxcr2-/- or Ccr1-/-Cxcr2-/- mice. Myeloid cells that express MMP2, MMP9 and VEGF were accumulated around both types of tumors through CCR1- and CXCR2-mediated pathways. Mice reconstituted with Ccr1-/-Cxcr2-/- BM exhibited the strongest suppression of tumor growth and liver metastasis compared with other three groups. Depletion of CCR1+CXCR2+ myeloid cells led to a higher frequency of CD8+ T cells, whereas the numbers of Ly6G+ neutrophils, FOXP3+ Treg cells and CD31+ endothelial cells were significantly decreased. Furthermore, treatment with a neutralizing anti-CCR1 mAb to mice reconstituted with Cxcr2-/- BM significantly suppressed tumor growth and liver metastasis. Dual blockade of CCR1 and CXCR2 pathways in myeloid cells could be an effective therapy against colorectal cancer.

GNUV201, a novel human/mouse cross-reactive and low pH-selective anti-PD-1 monoclonal antibody for cancer immunotherapy

BackgroundSeveral PD-1 antibodies approved as anti-cancer therapies work by blocking the interaction of PD-1 with its ligand PD-L1, thus restoring anti-cancer T cell activities. These PD-1 antibodies lack inter-species cross-reactivity, necessitating surrogate antibodies for preclinical studies, which may limit the predictability and translatability of the studies.ResultsTo overcome this limitation, we have developed an inter-species cross-reactive PD-1 antibody, GNUV201, by utilizing an enhanced diversity mouse platform (SHINE MOUSE™). GNUV201 equally binds to human PD-1 and mouse PD-1, equally inhibits the binding of human PD-1/PD-L1 and mouse PD-1/PD-L1, and effectively suppresses tumor growth in syngeneic mouse models. The epitope of GNUV201 mapped to the “FG loop” of hPD-1, distinct from those of Keytruda® (“C’D loop”) and Opdivo® (N-term). Notably, the structural feature where the protruding epitope loop fits into GNUV201’s binding pocket supports the enhanced binding affinity due to slower dissociation (8.7 times slower than Keytruda®). Furthermore, GNUV201 shows a stronger binding affinity at pH 6.0 (5.6 times strong than at pH 7.4), which mimics the hypoxic and acidic tumor microenvironment (TME). This phenomenon is not observed with marketed antibodies (Keytruda®, Opdivo®), implying that GNUV201 achieves more selective binding to and better occupancy on PD-1 in the TME.ConclusionsIn summary, GNUV201 exhibited enhanced affinity for PD-1 with slow dissociation and preferential binding in TME-mimicking low pH. Human/monkey/mouse inter-species cross-reactivity of GNUV201 could enable more predictable and translatable efficacy and toxicity preclinical studies. These results suggest that GNUV201 could be an ideal antibody candidate for anti-cancer drug development.

METTL14 regulates CD8+T-cell activation and immune responses to anti‐PD‐1 therapy in lung cancer

BackgroundN6-methyladenosine (m6A) modification plays an important role in lung cancer. However, methyltransferase-like 14 (METTL14), which serves as the main component of the m6A complex, has been less reported to be involved in the immune microenvironment of lung cancer. This study aimed to analyze the relationship between METTL14 and the immune checkpoint inhibitor programmed death receptor 1 (PD-1) in lung cancer.MethodsCCK-8, colony formation, transwell, wound healing, and flow cytometry assays were performed to explore the role of METTL14 in lung cancer progression in vitro. Furthermore, syngeneic model mice were treated with sh-METTL14 andan anti-PD-1 antibody to observe the effect of METTL14 on immunotherapy. Flow cytometry and immunohistochemical (IHC) staining were used to detect CD8 expression. RIP and MeRIP were performed to assess the relationship between METTL14 and HSD17B6. LLC cells and activated mouse PBMCs were cocultured in vitro to mimic immune cell infiltration in the tumor microenvironment. ELISA was used to detect IFN-γ and TNF-α levels.ResultsThe online database GEPIA showed that high METTL14 expression indicated a poor prognosis in patients with lung cancer. In vitro assays suggested that METTL14 knockdown suppressed lung cancer progression. In vivo assays revealed that METTL14 knockdown inhibited tumor growth and enhanced the response to PD-1 immunotherapy. Furthermore, METTL14 knockdown enhanced CD8+T-cell activation and infiltration. More importantly, METTL14 knockdown increased the stability of HSD17B6 mRNA by reducing its m6A methylation. In addition, HSD17B6 overexpression promoted the activation of CD8+ T cells.ConclusionThe disruption of METTL14 contributed to CD8+T-cell activation and the immunotherapy response to PD-1 via m6A modification of HSD17B6, thereby suppressing lung cancer progression.

IRX4204 Induces Senescence and Cell Death in HER2-positive Breast Cancer and Synergizes with Anti-HER2 Therapy.

Rexinoids, agonists of nuclear retinoid X receptor (RXR), have been used for the treatment of cancers and are well-tolerated in both animals and humans. However, the usefulness of rexinoids in treatment of breast cancer remains unknown. This study examines the efficacy of IRX4204, a highly specific rexinoid, in breast cancer cell lines and preclinical models to identify a biomarker for response and potential mechanism of action. IRX4204 effects on breast cancer cell growth and viability were determined using cell lines, syngeneic mouse models and primary PDX tumors. In vitro assays of cell cycle, apoptosis, senescence, and lipid metabolism were used to uncover a potential mechanism of action. Standard anti-HER2 therapies were screened in combination with IRX4204 on a panel of breast cancer cell lines to determine drug synergy. IRX4204 significantly inhibits the growth of HER2-positive breast cancer cell lines, including trastuzumab and lapatinib resistant JIMT-1 and HCC1954. Treatment with IRX4204 reduced tumor growth rate in the MMTV-ErbB2 mouse and HER2-positive PDX model by 49% and 44% respectively. Mechanistic studies revealed IRX4204 modulates lipid metabolism and induces senescence of HER2-positive cells. In addition, IRX4204 demonstrates additivity and synergy with HER2 targeted monoclonal antibodies, tyrosine kinase inhibitors, and antibody drug conjugates. These findings identify HER2 as a biomarker for IRX4204 treatment response and demonstrate a novel use of RXR agonists to synergize with current anti-HER2 therapies. Furthermore, our results suggest that RXR agonists can be useful for the treatment of anti-HER2 resistant and metastatic HER2-positive breast cancer.

Abstract PO3-26-08: Genetic modification of tumor associated macrophages using CRISPR lipid nanoparticle platform altered tumor microenvironment and induced cancer cell elimination

Abstract More than half of patients with stage IV breast cancer will eventually develop liver metastasis. Due to their nature, liver metastases are hard to treat, and the five-year survival rate for stage IV breast cancer is very low (~11%). In the recent years, vital roles macrophages play in cancer progression and immune evasion have become increasingly clear, making them an appealing target for therapeutic intervention. As one of the most abundant cell populations recruited to the tumor microenvironment, tumor-associated macrophages (TAM) are typically immunosuppressive Arg1+ M2-like macrophages, which support cancer growth, metastasis progression, and drug resistance. Numerous studies, including from our laboratory, have shown that reversal of the macrophage phenotype to the classical inflammatory CD80+ M1-like macrophages is very promising for cancer eradication. In this study, we used CRISPR loaded into lipid nanoparticles (CRISPR-LNP) to modulate macrophage polarization in breast cancer metastasis in vivo. We hypothesize that CRISPR-mediated gene editing through lipid nanoparticle (LNP) delivery of CRISPR enzyme and target gRNA (CRISPR-LNP) is a highly effective and safe approach. Using our CRISPR-LNP platform, we screened multiple genes with significant roles in macrophage polarization and found that targeting the RICTOR gene alone enabled reprograming of pro-tumorigenic M2 TAMs to anti-tumorigenic M1 phenotype in vitro and in vivo. We administered the CRISPR-LNP targeting RICTOR (CRISPR-RIC-LNP) on a syngeneic liver metastasis mouse model via intravenous injection. Single dose studies were conducted to determine biodistribution, treatment efficacy, and shift in immune landscape of the tumor after treatment via immunofluorescence staining, Imaging Mass Cytometry, and single cell RNA sequencing. A spatial map was built using digital pathology analysis for investigation of the interactions amongst immune and tumor cells within the tumor immune microenvironment. In 4T1 breast cancer liver metastatic model, we observed that CRISPR-RIC-LNP treatment: (i) accumulated in the liver metastases lesions in vivo 24 hours after injection. ~0.5% of total injected dose, which corresponds to ~75% of fluorescence from the whole body at 24h time point was confined only in the metastatic regions; (ii) led to modest increase of macrophage number, and (iii) significant shift of macrophage phenotype from immunosuppressive, pro-tumorigenic Arg1+, CD206+ macrophages to inflammatory, anti-tumor CD80+ phenotype (from ~5 to ~9% of total cell in the tumor lesions) 24 hours after injection; (iv) reduced the cancer cell number by almost 80%; and (v) dramatically reduced exhausted T-cell and regulatory T-cell population, as determined by single cell RNA sequencing analysis. We plan to validate our preliminary finding with flow cytometry analysis of metastatic lesions, and evalute the efficacy of CRISPR-RIC-LNP as single and in combinatorial therapies in survival studies. These data showed that reprograming macrophages from cancer-supporting to cancer-eliminating phenotype is a promising tool to suppress cancer cell growth and increase the presence of cytotoxic CD8+ T-cells. We propose that this approach can be efficiently used as single therapy or as combination with immune checkpoint inhibitors to obtain synergistic effects and efficiently eradicate cancer cells. We will assess the safety, optimize the accumulation in TME, and devise methods to limit exposure to healthy cells. In the future, we also aim to expand scope of our candidate therapy as therapeutics for other types of breast cancer metastases. Citation Format: Fransisca Leonard, Tyler Galbraith, Nourhan Abdelfattah, Arturas Ziemys, Chihiro Hashimoto, Harlan Cook, Yitian Xu, Shu-Hsia Chen, Roberto Rosato, Kyuson Yun. Genetic modification of tumor associated macrophages using CRISPR lipid nanoparticle platform altered tumor microenvironment and induced cancer cell elimination [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-26-08.

Abstract PO2-25-06: A hybrid breast cancer/mesenchymal stem cell population increases distant metastasis and chemoresistance of metaplastic breast carcinomas

Abstract Background: Metaplastic breast carcinoma is a heterogenous, highly aggressive and chemoresistant subtype of triple negative breast cancer (TNBC) with histological evidence of deregulated differentiation towards non-glandular components including spindle, squamous, and sarcomatoid. The role of the tumor microenvironment in the metastatic behavior and chemotherapy response of metaplastic carcinomas needs investigation to improve patient outcomes. Our lab has generated a model of spindle metaplastic breast carcinomas based on mammary epithelial cell-specific CCN6 knockout (MMTV-Cre;Ccn6fl/fl or CCN6KO). We have recently shown that a subset of TNBC cells engulf mesenchymal stem/stromal cells (MSC) generating a hybrid cell population with metastatic ability, which is detected in breast cancer tissue samples. In this study, we tested the hypothesis that hybrid cells may promote chemoresistant distant metastasis in spindle metaplastic breast carcinomas and that the tumor immune microenvironment may play a role in this function. Methods: Luc-GFP-CCN6KO cells were cultured with DsRED-MSCs for 7 days, yielding &amp;gt;90% of hybrid cells detected by flow cytometry. Hybrids or CCN6KO single cultures (1x10(5) cells) were injected intracardially in FVB/NJ syngeneic immune competent mice (n=20 mice per group). Four days after injection, each group was treated with Paclitaxel (PTX, 10 mg/kg twice a week, i.p.) or PBS control. The metastatic burden was evaluated by bioluminescence imaging twice a week. Mice were euthanized 23 days after heart injections, followed by necropsy and histopathological evaluation of the harvested organs. Results: All mice injected with single cultures of CCN6KO cells or hybrid cells formed distant metastasis. CCN6KO cells were highly invasive and spread to the lungs and lymph nodes in the chest,. We noted that 5 of 20 (25%) mice injected with hybrid cells showed metastasis in the liver (n=3 mice), pancreas (n=1), and abdominal lymph nodes (n=1). PTX treatment significantly reduced metastatic burden in mice injected with CCN6KO single cultures compared to vehicle (p=0.035) but had no effect on the metastatic burden of hybrid cells. Histologically, while the metastasis from CCN6KO cells and hybrids showed similar pathological features, consisting of spindle cancer cells with high grade nuclei and &amp;gt;10 mitoses/10 high power fields, hybrid-derived tumors had abundant infiltration by immune cells, mainly neutrophils, compared to single culture tumors. Conclusion: Our data show that hybrid cancer cell/MSC derived from CCN6KO spindle metaplastic carcinomas have higher ability to disseminate to distant organs, especially in the abdomen, compared to cancer cells. Our data show that hybrid cell-derived metastases have increased immune cell infiltration. We provide evidence that hybrid cells enhance Paclitaxel resistance in a mouse model of metaplastic breast carcinoma. Ongoing studies are aimed at elucidating the role of hybrids in the immune microenvironment of metaplastic breast carcinoma and clinical significance. Citation Format: Ahmad Eido, Maria Gonzalez, Celina Kleer. A hybrid breast cancer/mesenchymal stem cell population increases distant metastasis and chemoresistance of metaplastic breast carcinomas [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-25-06.

Abstract PO1-25-01: LMTK3 promotes macrophage M2 polarisation and suppresses monocyte infiltration in breast cancer by altering the proteomic cargo of exosomes

Abstract Background: Breast cancer (BC) is a leading cause of cancer death and despite numerous treatment advancements, metastatic BC is incurable. Extracellular vesicles (EVs) are lipid-delimited particles released from cells which contain bioactive protein and nucleic acid cargo. Numerous studies have shown their importance in facilitating communication between cancer cells and the tumour microenvironment, implicating them as mediators of many aspects of carcinogenesis. EV-mediated communication between BC and the innate immune system plays a crucial role in BC progression, metastasis and the maintenance of an immunosuppressive microenvironment. LMTK3 overexpression in BC promotes therapy resistance, disease progression and invasion and metastasis, leading to a poorer prognosis in patients. Analysis of deconvoluted TCGA data indicated that LMTK3 overexpression also correlates with exclusion of M1 macrophages from the tumour microenvironment. This study aimed to explore the role of LMTK3 in EV-mediated communication between BC and immune cells. Methods EVs were collected from conditioned media of wild-type and LMTK3-overexpressing BC cells through differential ultracentrifugation, then characterised by transmission electron microscopy, nanoparticle tracking analysis, quantitative mass spectrometry and Western blotting. Monocytes were treated with EVs and proteomic and phosphoproteomic changes were analysed. 3D coculture models of BC and immune cells were used to assess monocyte and macrophage infiltration, polarisation and anti-cancer activity. Immunofluorescence and flow cytometry was used to assess macrophage polarisation. Bioinformatic analysis of TCGA datasets validated the findings in a clinical setting and the data was also validated in vivo, using syngeneic immunocompetent mouse models. Results LMTK3 overexpression in BC cells promotes the upregulation of a subpopulation of large (80-150nm) CD81+ EVs which are significantly enriched in oncogenic and immunoregulatory proteins, including LDHB, ARHGEF15, PSAT1 and HIC2. EVs from LMTK3-overexpressing but not control BC cells strongly reduce the ability of monocytes to infiltrate BC tumour spheroids and migrate through Matrigel. Proteomics analysis of the monocytes showed an enrichment in M2 polarisation proteins and a downregulation of proteins essential for migration, including S100A8, KLF13 and TRPC3. LMTK3 overexpression in BC also promotes M2 polarisation and exclusion of M1 macrophages in vivo. Summary LMTK3 overexpression in BC correlates with poor prognosis and reduced overall survival. We hypothesise that this is, in part, due to monocyte M2 polarisation and exclusion of M1 macrophages from the TME, mediated by LMTK3-dependent alterations in EV cargo, contributing to tumour immune evasion. Citation Format: Mark Samuels, William Jones, Charlotte Turner, Georgios Giamas. LMTK3 promotes macrophage M2 polarisation and suppresses monocyte infiltration in breast cancer by altering the proteomic cargo of exosomes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-01.

Abstract PO5-24-12: IRX5010, a Highly Selective RARγ Nuclear Receptor Agonist Compound Inhibits In Vivo Growth of Murine EMT-6 Triple Negative Breast Cancer and Human JIMT-1Her2+ Breast Cancer By Induction of Tumor-Infiltrating Effector Memory T-cells

Abstract Research Objectives and Rationale: Previously, RARγ agonism has been shown to play an essential role in CD8 T-cell-mediated immunity to infectious pathogens. However, we found no previous reports on effects of RARγ agonists on in vivo tumor growth of triple negative or Her2+ breast cancers. Here we explored whether RARγ could play a critical role in T-cell-mediated immunity in these breast cancers, using potent highly selective novel RARγ agonist compounds. Experimental methods and results: We synthesized and screened compounds for selective transactivation of RAR γ in reporter assays. Three highly potent compounds, demonstrating highly selective RARγ transactivation at less than one nanomolar concentrations, were selected for further evaluation of potential anti-cancer activities in vitro. None of the three evaluated compounds demonstrated significant inhibitory effects on in vitro growth of EMT-6 triple negative breast cancer cells at concentrations of 20-100 millimolar, indicating that the three compounds have insignificant direct inhibitory effects on EMT-6 cancer cell growth in vitro. We then examined the effects of one of the compounds in vivo in a syngeneic mouse model of EMT-6 triple negative breast cancer. Treatment with the most potent of the three compounds, IRX5010, demonstrated dose response inhibition of in vivo growth of EMT-6 tumors. Inhibition of tumor growth at day 17 relative to vehicle was 59% and 69% respectively in mice treated with IRX5010 at 10 or 25 mg/kg/day. A cohort treated with 50 mg/kg/day was sacrificed early on day 12 due to excessive weight loss and deaths of some of the animals. This cohort experienced a 73% inhibition of tumor growth relative to vehicle. Gross necropsies of animals in the 25 and 50 mg/kg/day dose groups did not show observable pathologic findings in any major organ. Flow cytometry was performed on freshly excised tumors on the day of termination of the in-life portion of the study in the 10 and 25 mg/kg/day dose groups. Tumors from mice treated with IRX5010 had increased intratumoral numbers of Total CD45+ cells; Total CD3+, Total CD4+, and Total CD8+ T-cells per mg of tumor tissue, relative to vehicle controls. IRX5010 treated mice also had increased intratumoral CD4+IL-17+ T-helper cells; and increased CD4+CD62L- and CD8+CD62L- T-effector memory T-cells per mg of tumor tissue relative to vehicle-treated control mice. We also conducted an experiment of treatment with IRX5010 in mice transplanted with human immune cells (to provide a human immune system) to examine its effects on xenografted human Her2+ JIMT-1 cancer cells. In this model, treatment with IRX5010 at 10 mg/kg/day resulted in a 17% inhibition of tumor growth relative to vehicle control over 26 days of treatment. Flow cytometric analysis showed that tumors from the IRX5010 treated mice contained increased numbers per mg of tumor tissue of intratumoral human CD8+ T-cells, most of which were CD8+ CD7- CD45RA+ terminally differentiated effector memory phenotype T-cells. Conclusions: Treatment with IRX5010, a potent and highly selective RARγ nuclear receptor agonist compound demonstrated substantial treatment effects relative to vehicle control on inhibition of growth of murine triple negative breast cancer in vivo, despite having essentially no growth inhibitory effect on these cells in vitro. Treatment of xenografted human Her2+ tumors in mice with humanized immune systems resulted in a moderate inhibition of Her2+ breast cancer growth relative to vehicle controls. In both models, treatment with IRX5010 induced tumor infiltrating T-lymphocytes of largely effector memory phenotypes, demonstrating an effect on the tumor immune microenvironment. These data identify RARγ and RARγ agonists as a new treatment target and novel treatment modality for breast cancers, especially for the highly unmet need for new and better treatments for triple negative breast cancer. Citation Format: Martin Sanders, Mary Topalovski, Vidyasagar Vuligonda. IRX5010, a Highly Selective RARγ Nuclear Receptor Agonist Compound Inhibits In Vivo Growth of Murine EMT-6 Triple Negative Breast Cancer and Human JIMT-1Her2+ Breast Cancer By Induction of Tumor-Infiltrating Effector Memory T-cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-12.

Abstract PS07-06: A novel citrullinated-ENO1 peptide-based vaccine for triple-negative breast cancer prevention

Abstract Purpose: To evaluate the merits of a novel citrullinated-enolase 1 (ENO1) peptide-based vaccine for inducing an effective response against triple negative breast cancer (TNBC). Background: Targets for cancer vaccines have included mutated proteins and peptides, as well as proteins that are overexpressed in cancer but otherwise ubiquitous in their structure. However, protein modifications other than mutation may also induce an immune response. Several lines of evidence have demonstrated a role for the posttranslational modification (PTM) citrullination as inducing immunogenicity. Dysregulated protein citrullination by peptidyl arginine deiminases (PADI) has been associated with autoimmune diseases and is currently being explored for its relevance to cancer. Previously, our group established PADI2, which catalyzes the PTM from arginine to citrulline, to be highly expressed in TNBC compared to normal mammary and other tissues. We observed that PADI2-associated protein citrullination promotes antigenicity. Importantly, we demonstrated that protein citrullination, non-occurring in DNA and RNA-based vaccines, is highly cancer specific. On the basis of these findings, we developed a novel vaccine based on cancer-selective citrullinated-enolase 1 peptides (cit-ENO1) that were identified from mass spectrometry surfaceome and immunopeptidome data from breast cancer cells. We assessed the extent to which a citENO1 peptide based vaccine elicits a potent host response and attenuates tumor development in a syngeneic mouse model of TNBC. Design: To confirm that the cit-ENO1 peptide vaccine elicited an immune response, we immunized female B6129SF1/J mice with 10nmol cit-ENO1 peptides or 10nmol of corresponding unmodified peptides as the antigen, plus the TLR3 agonist poly I:C as an adjuvant (10mg), subcutaneously once a week, for three weeks. Saline or the adjuvant alone were used as controls. One week post the last round of immunization, draining lymph nodes (LNs) and immunized skin were harvested, and T cell phenotypes evaluated using flow-cytometry. To assess anti-cancer efficacy, female B6129SF1/J mice were immunized with cit-ENO1 peptides or unmodified peptides with poly I:C as an adjuvant followed by orthotopic implantation with murine BRCA1co/co; MMTV-Cre; p53+/- TNBC cells (1.5 x 106) and tumor growth as well as overall survival were assessed. Results: We observed that the cit-ENO1 peptide vaccination induced a statistically significantly greater percentage of activated CD8+ PD-1+ effector T cells as well as CD8+ CD44+ CD62L- effector memory T cells in the LNs compared with either the control groups or the unmodified ENO1 peptide. CD8+ PD-1+ T-cells were also found to be significantly elevated in immunized skin of cit-ENO1 vaccinated mice. Both cit-ENO1 and unmodified ENO1 vaccine increased CD4+ PD-1+ T cells from the LNs and immunized skin. Remarkably, the cit-ENO1 vaccine but not the unmodified-ENO1 vaccine significantly delayed tumor growth and markedly improved overall survival compared to respective controls (Logrank test for trend 2-sided p&amp;lt; 0.05). Conclusion: We have developed a novel vaccine based on cancer-selective cit-ENO1 peptides that stimulates a potent anti-cancer immune response and significantly delays tumor initiation and progression in a preclinical, immune competent mouse model of TNBC. Importantly, the anti-cancer effects were not observed with corresponding unmodified ENO1 peptides. Citation Format: Ricardo Leon Letelier, Hiroyuki Katayama, Yihui Chen, Yinning Cai, Fuchung Hsiao, Banu Arun, Samir Hanash. A novel citrullinated-ENO1 peptide-based vaccine for triple-negative breast cancer prevention [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS07-06.

Abstract PO5-25-01: Treating liver metastases by reversing cell competition between metastatic cancer cells and hepatocytes

Abstract Background: Although breast cancer liver metastases (BCLM) are common events, with 40-50% of patients with metastatic breast cancer developing liver metastases, their development indicates poor prognosis. Patients with BCLM have a 5-year survival rate of 8%. Specific treatments for liver metastases are virtually non-existent, partly because the key events of early liver metastasis formation remain unknown. The liver is a dense organ, and metastatic breast cancer cells (MCC) must clear space among hepatocytes, the primary cell population in the liver, during metastatic formation. Cell competition is a developmentally conserved process that explains how organs or primary tumors form when cells within those tissues have varying fitness and compete with one another. This competition can result in the survival and expansion of one cell population, and the induction of cell death through apoptosis in the other. We hypothesized that MCC use cell competition with hepatocytes to create space and expand within the liver. Objectives: In this study, we tested the hypothesis that MCC use cell competition with hepatocytes to create space and expand within the liver, resulting in hepatocyte apoptosis. Methods: We develop new in-vitro and mouse lineage-traced models to assay cell competition between MCC and hepatocytes, initially using balb/c derived breast cancer (4T1) and hepatocyte (H2.35) cells and syngeneic mouse models of liver metastasis. These models directly capture MCC and hepatocyte interactions, and we can assess modes of cell competition, including apoptosis, through immunostaining. Because prior literature suggests that the developing mammary gland informs both cell competition and metastasis, we next performed a bioinformatics screen to identify potential signals that contribute to cell competition between MCC and hepatocytes. We generated a new single-cell RNA-seq dataset that incorporates information from the developing mammary gland and breast cancer cells. This dataset includes 3264 primary breast cancer cells across 3 models of aggressive breast cancers, encompassing HER2+ and TNBC subtypes, as well as 526 embryonic mammary gland cells. Using this platform, we identify genes enriched in both breast cancer and the developing mammary gland. Next, we validate genes by testing for their high expression in MCC when compared to hepatocytes. Then, we determine the impact of target genes on cell competition in our models. Results/Discussion: Our models demonstrate that MCC use cell competition to create space in the liver by inducing apoptosis in neighboring hepatocytes. MCC induce apoptosis in neighboring hepatocytes through cell competition in in-vitro co-cultures by 11.7-fold compared to MCC apoptosis. Hydrodynamic tail-vein engraftment in NOD-scid-gamma mice livers with MCC also resulted in increased neighboring hepatocyte apoptosis. Using our bioinformatics screen, we identified 5 potential targets that could drive breast cancer metastasis and cell competition. Using our competition models, perturbation of SerpinE2, a protease inhibitor which was one of the top gene targets, successfully modulated competition between MCC and hepatocytes. Knock-out of SerpinE2 in MCC did not affect their viability or growth but resulted in MCC apoptosis adjacent to hepatocyte neighbors by 16.9-fold when compared to wild type conditions. Further, co-culture of MCC that lacked SerpinE2 with hepatocytes resulted in decreased hepatocyte apoptosis by 2.3-fold compared to hepatocytes co-cultured with wild-type (WT) MCC. These findings suggest that hepatocytes cannot outcompete or kill WT MCC, but they can outcompete and kill MCC that lack SerpinE2. Future work focused on the perturbation of SerpinE2 may lead to novel therapies for patients at risk for or with BCLM. Our findings suggest that restoring the competitiveness of the liver parenchyma could be a new treatment modality for treating liver metastases. Citation Format: Katherine Lake, Sakshi Mohta, Clayton Smith, Venkata Repaka, Lily Xu, Kaitlyn Saunders, Vrushali Pandit, Emily Goff, Christine Zhang, Jacob Pena, Christine Hodgdon, Julia Maues, Elizabeth Chen, Isaac Chan. Treating liver metastases by reversing cell competition between metastatic cancer cells and hepatocytes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-25-01.

Targeted desialylation and cytolysis of tumour cells by fusing a sialidase to a bispecific T-cell engager.

Bispecific T-cell engagers (BiTEs) bring together tumour cells and cytotoxic T cells by binding to specific cell-surface tumour antigens and T-cell receptors, and have been clinically successful for the treatment of B-cell malignancies. Here we show that a BiTE-sialidase fusion protein enhances the susceptibility of solid tumours to BiTE-mediated cytolysis of tumour cells via targeted desialylation-that is, the removal of terminal sialic acid residues on glycans-at the BiTE-induced T-cell-tumour-cell interface. In xenograft and syngeneic mouse models of leukaemia and of melanoma and breast cancer, and compared with the parental BiTE molecules, targeted desialylation via the BiTE-sialidase fusion proteins enhanced the formation of immunological synapses, T-cell activation and T-cell-mediated tumour-cell cytolysis in the presence of the target antigen. The targeted desialylation of tumour cells may enhance the potency of therapies relying on T-cell engagers.

Supplementation of Spirulina (= Arthrospira) Platensis Induces Immunosuppression through Increasing T-regulatory Cells in a Syngeneic Mouse Model of Breast Cancer

Supplementation of Spirulina (= Arthrospira) Platensis Induces Immunosuppression through Increasing T-regulatory Cells in a Syngeneic Mouse Model of Breast Cancer

A new MVA ancestor-derived oncolytic vaccinia virus induces immunogenic tumor cell death and robust antitumor immune responses

Vaccinia viruses (VACV) are versatile therapeutic agents and different features of various VACV strains allow for a broad range of therapeutic applications. Modified Vaccinia virus Ankara (MVA) is a particularly altered VACV strain that is highly immunogenic, incapable of replicating in mammalian hosts, and broadly used as a safe vector for vaccination. Alternatively, Western Reserve (WR) or Copenhagen (Cop) are VACV strains that efficiently replicate in cancer cells and therefore are used to develop oncolytic viruses. However, the immune evasion capacity of WR or Cop hinders their ability to elicit antitumor immune responses, which is crucial for efficacy in the clinic. Here, we describe a new VACV strain named Immune-Oncolytic Vaccinia virus Ankara (IOVA), which combines efficient replication in cancer cells with induction of immunogenic tumor cell death (ICD). IOVA was engineered from an MVA ancestor and shows superior cytotoxicity in tumor cells. In addition, the IOVA genome incorporates mutations that lead to massive fusogenesis of tumor cells, which contributes to improved antitumor effects. In syngeneic mouse tumor models, induction of ICD results in robust antitumor immunity directed against tumor neo-epitopes and eradication of large established tumors. These data present IOVA as an improved immunotherapeutic oncolytic vector.

Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

ObjectivesEpithelial ovarian cancer (EOC) is considered to be a prevalent female malignancy with both high incidence and mortality. It is reported that RNA-binding protein 3 (RBMS3) executives a tumor suppressor function in different cancers. This investigation was designed to examine the expression of RBMS3 in epithelial ovarian cancer, the effects on EOC cells, and its connection to immune cells that infiltrate tumors in the EOC microenvironment. MethodsThe expression levels of RBMS3 in EOC tissues as well as their correlations with immune cell infiltration and clinical outcome were examined using bioinformatics approaches. Western blotting as well as immunohistochemistry were carried out to determine the protein levels in EOC tissues. In addition, qRT-PCR was employed to look at the expression of the mRNA. The role of RBMS3 in EOC cells was investigated, and an RBMS3 lentiviral vector was developed. The effects of RBMS3 on subcutaneous tumor development, the proliferation protein Ki-67, the tumor angiogenesis indicator CD31, and its function in controlling the tumor immune microenvironment were evaluated by in vivo tests. ResultsThere was a considerable decrease in RBMS3 expression in EOC tissues, which was linked to a poor prognosis for patients and the infiltration of multiple immune cell. Given immunohistochemical studies, tissues with increased RBMS3 expression had decreased markers of myeloid-derived suppressor cells, regulatory T cells, and M2 macrophages, whereas M1 macrophage markers were elevated. RBMS3 appears to suppress the capabilities of proliferating, invading, and migrating in EOC cells according to in vitro tests, whereas tumors overexpressing RBMS3 developed more slowly in syngeneic mouse models. The overexpression of RBMS3 led to a decline in the levels of Ki-67 protein and CD31. Additionally, it showed a negatively correlation with markers of regulatory T cell, myeloid-derived suppressor cell, and M2 macrophage but a positive correlation with markers of M1 macrophage. ConclusionsThe findings revealed that elevated RBMS3 expression plays a tumor suppressor role in EOC and was connected to patient survival in EOC. The studies conducted in vitro and in vivo demonstrated a link between RBMS3 expression and the infiltration of certain immune cells, indicating a function for RBMS3 in the immunosuppressive tumor microenvironment and its promising efficiency as a novel target for immunotherapy against EOC.