4T1 Murine Breast Cancer Research Articles

Establishing and Characterizing Chemotherapy-Induced Immunological Dormant Tumor Cell Lines.

Chemotherapy, together with radiotherapy, targeted therapies, and immunotherapy, is the main option to treat cancer patients in neoadjuvant/adjuvant setting to reduce the risk of disease progression and metastasis formation from disseminated tumor cells. Cancer cells that survived chemotherapy treatment may emerge with novel characteristics, one of which is the ability to stimulate the native and adaptive immune systems. Models allowing the characterization of chemotherapy-induced tumor cell plasticity and induction of immune response or adaptation are needed to identify novel mechanisms and devise novel therapeutic strategies to prevent relapses. Here we describe a protocol for selecting chemotherapy-resistant cancer cells and testing the in vivo effects on the local and systemic immune responses. While originally developed to characterize the effects of methotrexate and doxorubicin on murine 4T1 breast cancer cells and the relative immune response, the method can be broadened to other chemotherapies and syngeneic cancer models.

Characterization and anti-tumor activities of polysaccharide isolated from Brassica rapa L. via activation of macrophages through TLR2-and TLR4-Dependent pathways

We have previously shown the immunostimulatory effects by Nozawana (Brassica rapa L.). In this report, we determined the characteristics of Nozawana polysaccharide (NPS) and evaluated the immunomodulatory effects and anti-tumor activity of NPS mediated by macrophage activation. The molecular weight of NPS was determined by gel filtration chromatography with an average molecular weight of approximately 100.6 kDa. HPLC analysis showed that NPS contained glucose, galacturonic acid, galactose, and arabinose. NPS increased cytokine and nitric oxide (NO) production by macrophages in a Toll-like receptor (TLR)2 and TLR4-dependent manner. Furthermore, NPS induced apoptosis significantly against 4T1 murine breast cancer cells cultured in conditioned medium from NPS-treated macrophages through tumor necrosis factor-α. In tumor-bearing mouse model, tumor growth was significantly reduced in NPS-treated mice compared with control mice. These results support the potential use of NPS as an immunotherapeutic material found in health food products.

The triple-drug combination DBDx enhances the antitumor efficacy of PD-1 antibody associated with Treg modulation.

This study investigated the antitumor efficacy of programmed cell death protein-1 (PD-1) antibody and DBDx, a triple-drug combination of dipyridamole, bestatin, and dexamethasone, and their related immunomodulation. Mouse melanoma B16, mouse Lewis lung carcinoma, and mouse breast carcinoma 4T1 were used for evaluating the in vivo therapeutic efficacy of DBDx, PD-1 antibody, and their combination. The peripheral blood and tumor tissues of 4T1 tumor-bearing mice were collected to analyze regulatory T cells and measured using flow cytometry. The combination of PD-1 antibody and DBDx enhanced the therapeutic efficacy against B16 melanoma. The suppression of tumor growth by PD-1 antibody and DBDx was more significant than that by anti-PD-1 monotherapy. The tumor growth inhibition rates of PD-1 antibody, DBDx, and their combination were 54.0%, 72.4%, and 83.1%, respectively, suggesting a synergistic effect as determined by the coefficient of drug interaction. No significant changes were found in the body weights in all the above groups, indicating that the treated mice tolerated the applied drug doses. Similarly, enhanced therapeutic efficacy of the PD-1 antibody and DBDx combination was observed in murine Lewis lung carcinoma and 4T1 breast cancer models. In 4T1 breast cancer-bearing mice, the immunotherapy-related changes in lymphocytes in peripheral blood and tumor microenvironment were evaluated with flow cytometry. Compared with anti-PD-1 monotherapy, peripheral blood and tumor-infiltrating lymphocytes were found a lower ratio of regulatory T cell (Treg) subset cells and a higher ratio of CD8+/Treg cells. The combination of PD-1 antibody and DBDx could achieve enhanced therapeutic antitumor efficacy than anti-PD-1 monotherapy, suggesting potential for using the triple-drug combination DBDx in cancer immunotherapy.

Abstract C079: Comprehensive immunophenotypic profiling sheds light on the dynamic interplay between immune cells in the 4T1 breast cancer model upon anti-PD1 and anti-CTLA4 immunotherapy

Abstract Immuno-oncology has revolutionized cancer treatment by harnessing the immune system to target and eliminate tumor cells. In this study, we employed a combination of fluorescence-activated cell sorting (FACS) and Cytek full spectrum flow cytometry to investigate the impact of anti-PD1 (programmed cell death protein 1) and anti-CTLA4 (cytotoxic T-lymphocyte-associated protein 4) treatments on the 4T1 breast cancer model. We comprehensively analyzed 25 surface markers on immune cells to gain insights into the dynamic changes induced by immunotherapy. The 4T1 murine breast cancer model is known for its aggressive and metastatic nature, resembling triple-negative breast cancer in humans. We established a syngeneic orthotopic tumor graft of 4T1 cells in immunocompetent mice and treated them individually with anti-PD1 or anti-CTLA4 antibodies and compared the results to the respective isotype controls. Using FACS, we characterized immune cell populations within the tumor microenvironment and spleenocytes at multiple time points (before treatment, day 5, and day 17) during the treatment course. The Cytek full spectrum flow cytometry platform allowed simultaneous measurement of 25 surface markers, enabling a detailed analysis of immune cell phenotypes and functional states in a single experiment. Our findings revealed significant alterations in the immune landscape following anti-PD1 or anti-CTLA4 treatments. We observed an increase in tumor-infiltrating lymphocytes (TILs) and specifically CD4+ as well as CD8+ T cell infiltration within the tumor, accompanied by upregulation of T cell activation markers, such as CD69, CD38, CD44, PD1 and CD25. The immune cell composition within 4T1 tumors included CD4+ and CD8+ TILs, as well as increased populations of monocytes and macrophages. Additionally, a decrease in regulatory T cells (TRegs) was observed, indicating a potential restoration of immune balance. Moreover, F4/80+ CD206+ tumor-associated macrophages (TAMs) were identified in the 4T1 tumors vs. the spleen. This was associated with increased granzyme B production by CD8+ T cells and a shift towards an effector memory phenotype, suggesting improved cytotoxic function. To gain insights into spatial infiltration patterns of immune cells, we performed Immunohistochemistry (IHC) stainings of various immune cell markers in the 4T1 tumors. In line with the FACS results, we observed increased immune cell infiltration in tumors treated with anti-PD1 and anti-CTLA4 antibodies. Our comprehensive immunophenotypic analysis, utilizing Cytek full spectrum flow cytometry and IHC sheds light on the dynamic interplay between immune cells and the 4T1 breast cancer model during anti-PD1 and anti-CTLA4 immunotherapy. These findings provide valuable insights into the mechanisms underlying the therapeutic efficacy of immune checkpoint blockade in this aggressive cancer model. Citation Format: Christos Nikolaou, Simon Heller, Stefan Kaulfuss, Carlo Stresemann, Alexandra Eichten, Oliver von Ahsen, Martin Lange. Comprehensive immunophenotypic profiling sheds light on the dynamic interplay between immune cells in the 4T1 breast cancer model upon anti-PD1 and anti-CTLA4 immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C079.

Optimizing the administrated light dose during 5-ALA-mediated photodynamic therapy: Murine 4T1 breast cancer model.

Photodynamic therapy (PDT) is already used to treat many cancers, including breast cancer, the most common cancer in women worldwide. The destruction basis of this method is on produced singlet oxygen which is extremely reactive and is a major agent of tumor cell killing. The measurement of singlet oxygen produced within PDT is essential in predicting treatment outcomes and their optimization. This study aims to determine the optimal total light dose administered during PDT by calculating the singlet oxygen to facilitate the prediction of the treatment outcome in mice bearing 4T1 cell breast cancer. Monitoring the changes in photosensitizer fluorescence signals during PDT due to photobleaching can be one of the methods of determination of singlet oxygen generation in the PDT process. This study determined the oxygen singlet as a photodynamic dose from the three-dimensional Monte Carlo method and the photobleaching empirical dose constant. The photobleaching dose constant was established non-invasively by monitoring the invivo protoporphyrin IX (PpIX) fluorescence and photobleaching during PDT. The photobleaching dose constant (β) in J/cm2 was calculated using empirical fluorescence data. The invivo photobleaching dose constant of aminolevulinic acid was found to be 11.6 J/cm2 and based on this value, the optimal treatment light dose was estimated at 120 J/cm2 in mice bearing 4T1 breast cancer. It is concluded that information can be obtained regarding optimal treatment parameters by monitoring the invivo PpIX fluorescence and photobleaching during PDT.

The activation of SIRT1 by resveratrol reduces breast cancer metastasis to lung through inhibiting neutrophil extracellular traps

Neutrophil extracellular traps (NETs) play a crucial role in breast cancer metastasis. However, the therapeutic target of NETs in breast cancer metastasis is still unknown. Using a natural metabolite library and single-cell sequencing data analysis, we identified resveratrol (RES), a polyphenolic natural phytoalexin, and agonist of silent information regulator-1 (SIRT1) that suppressed NETs formation after cathepsin C (CTSC) treatment. In vivo, RES significantly hindered breast cancer metastasis in a murine orthotopic 4T1 breast cancer model. Serum levels of myeloperoxidase-DNA and neutrophil elastase-DNA in mouse breast cancer model were significantly lower after RES treatment. Correspondingly, the tumour infiltrated CD8+T cells in the lungs increased after the treatment. Mechanistically, RES targets SIRT1 in neutrophils and significantly inhibits the citrullination of histones H3, which is essential for chromatin decondensation and NETs formation. Furthermore, we identified that the NETs were suppressed by RES in bone marrow neutrophils after CTSC treatment, while specific deficiency of SIRT1 in neutrophils promoted NETs formation and breast cancer to lung metastasis. Thus, our results revealed that RES could be potentially identified as a viable therapeutic drug to prevent neutrophil cell death and breast cancer metastasis.

B-373 The Development of a Logic-Gate Probe to Investigate Hypoxia in Cancer Stem Cells

Abstract Background Cancer stem cells (CSCs) represent only a small fraction of the tumor population, yet they present a profound challenge to cancer treatment. CSCs contribute to tumor complexity via their unique ability to self-renew, develop into multiple lineages, and maintain an unchecked proliferation potential. To identify CSCs, the cytoprotective enzyme aldehyde dehydrogenase 1A1 (ALDH1A1) is a well-annotated biomarker of cancer stemness which is associated with a poor prognosis in patients with gastric and lung cancers when overexpressed. Within CSCs, ALDH1A1 functions to regulate oxidative stress by transforming biological aldehydes into their corresponding non-toxic carboxylic acid counterparts and further is linked to the retinaldehyde signaling pathway. Beyond this enzyme, oxygen availability within the tumor microenvironment (TME) can also influence stemness properties. Specifically, hypoxia, is required to maintain the CSC phenotype by prolonging the undifferentiated state. Methods To study the mechanistic underpinnings of complex relationship in vivo new technologies are needed as current approaches are insufficient. Indeed, probes have been developed to study ALDH1A1 and hypoxia in parallel, however, imaging approaches lack the requisite resolution to ensure they are reporting on the same CSC population. To this end, we have designed a powerful logic-gate probe that links the reporters of ALDH1A1 activity and oxygenation in a unique manner such that different readouts will result (color of light) depending on the TME environment. Results Our lab has previously developed a near-infrared hypoxia-sensing fluorescent probe that is fundamental to our current logic-gate design. This component has been validated in 4T1 murine breast cancer cells, to yield a 1.6-fold increase in ratiometric fluorescence response when incubated under hypoxic conditions as compared to its normoxic controls. Additionally, the ALDH1A1 sensing component has been validated via purified enzyme to yield an increase in bioluminescent signal enabling its use for future in vivo studies. Conclusion This technology has important clinical implications to understand the mechanistic underpinnings between oxygenation and cancer stemness. Further research is being conducted to further test the hypothesis that metformin (anticancer drug) kills cancer by altering oxygen metabolism which in turn depletes the CSC population. Furthermore, this probe has far-reaching implications in monitoring novel therapeutic cancer treatment.

Tumor cell-expressed lipolysis-stimulated lipoprotein receptor negatively regulates T-cell function.

Lipolysis-stimulated lipoprotein receptor (LSR) is known as a lipoprotein receptor. LSR is expressed in various solid tumors, including epithelial ovarian, gastric, and colon cancers. High LSR expression is significantly associated with poor prognosis, but its role in cancer has not been fully elucidated. LSR belongs to the Ig protein superfamily, which is conserved in B7 family. Here, we assessed LSR as a novel immune checkpoint molecule. We developed a novel anti-LSR antibody (#27-6 mF-18) that defects antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activity. The #27-6 mF-18 cross-reacts with both human and mouse LSR. We found that LSR was expressed on 4T1 murine breast cancer cell line. The #27-6 mF-18 exhibited antitumor effects against the 4T1 syngeneic tumor model, a poor immunogenic model refractory to treatment with anti-PD-1 or anti-CTLA-4 antibodies. Compared with control antibody-treated mice, mice treated with #27-6 mF-18 showed significantly increased numbers of CD8+ T cells and a ratio of activated CD8+ T cells infiltrated in the tumor tissue. This antitumor effect was abrogated by CD8+ T-cell depletion through anti-CD8 antibody treatment, indicating that LSR negatively regulates tumor immunity by repressing CD8+ T cells. These findings show that LSR negatively regulates T-cell immune activity. LSR targeting could provide immune checkpoint inhibitors for cancer immunotherapy.

Multiparametric chemical exchange saturation transfer MRI detects metabolic changes in breast cancer following immunotherapy

BackgroundWith metabolic alterations of the tumor microenvironment (TME) contributing to cancer progression, metastatic spread and response to targeted therapies, non-invasive and repetitive imaging of tumor metabolism is of major importance. The purpose of this study was to investigate whether multiparametric chemical exchange saturation transfer magnetic resonance imaging (CEST-MRI) allows to detect differences in the metabolic profiles of the TME in murine breast cancer models with divergent degrees of malignancy and to assess their response to immunotherapy.MethodsTumor characteristics of highly malignant 4T1 and low malignant 67NR murine breast cancer models were investigated, and their changes during tumor progression and immune checkpoint inhibitor (ICI) treatment were evaluated. For simultaneous analysis of different metabolites, multiparametric CEST-MRI with calculation of asymmetric magnetization transfer ratio (MTRasym) at 1.2 to 2.0 ppm for glucose-weighted, 2.0 ppm for creatine-weighted and 3.2 to 3.6 ppm for amide proton transfer- (APT-) weighted CEST contrast was conducted. Ex vivo validation of MRI results was achieved by 1H nuclear magnetic resonance spectroscopy, matrix-assisted laser desorption/ionization mass spectrometry imaging with laser postionization and immunohistochemistry.ResultsDuring tumor progression, the two tumor models showed divergent trends for all examined CEST contrasts: While glucose- and APT-weighted CEST contrast decreased and creatine-weighted CEST contrast increased over time in the 4T1 model, 67NR tumors exhibited increased glucose- and APT-weighted CEST contrast during disease progression, accompanied by decreased creatine-weighted CEST contrast. Already three days after treatment initiation, CEST contrasts captured response to ICI therapy in both tumor models.ConclusionMultiparametric CEST-MRI enables non-invasive assessment of metabolic signatures of the TME, allowing both for estimation of the degree of tumor malignancy and for assessment of early response to immune checkpoint inhibition.

Triple negative breast cancer metastasis is hindered by a peptide antagonist of F11R/JAM‑A protein

BackgroundThe F11R/JAM-A cell adhesion protein was examined as the therapeutic target in triple negative breast cancer (TNBC) with the use of the peptide antagonist to F11R/JAM-A, that previously inhibited the early stages of breast cancer metastasis in vitro.MethodsThe online in silico analysis was performed by TNMPlot, UALCAN, and KM plotter. The in vitro experiments were performed to verify the effect of peptide 4D (P4D) on human endothelial cell lines EA.hy926 and HMEC-1 as well as on human TNBC cell line MDA-MB-231. The cell morphology upon P4D treatment was verified by light microscopy, while the cell functions were assessed by colony forming assay, MTT cell viability assay, BrdU cell proliferation assay, and Transepithelial/Endothelial Electrical Resistance measurements. The in vivo experiments on 4T1 murine breast cancer model were followed by histopathological analysis and a series of quantitative analyses of murine tissues.ResultsBy in silico analysis we have found the elevated gene expression in breast cancer with particular emphasis on TNBC. The elevated F11R expression in TNBC was related with poorer survival prognosis. Peptide 4D has altered the morphology and increased the permeability of endothelial monolayers. The colony formation, viability, and proliferation of MDA-MB-231 cells were decreased. P4D inhibited the metastasis in 4T1 breast cancer murine model in a statistically significant manner that was demonstrated by the resampling bootstrap technique.ConclusionsThe P4D peptide antagonist to F11R/JAM-A is able to hinder the metastasis in TNBC. This assumption needs to be confirmed by additional 4T1 mouse model study performed on larger group size, before making the decision on human clinical trials.

HDACs alters negatively to the tumor immune microenvironment in gynecologic cancers

The role of histone deacetylases (HDACs) in the tumor immune microenvironment of gynecologic tumors remains unexplored. We integrated data from The Cancer Genome Atlas and Human Protein Atlas to examine HDAC expression in breast, cervical, ovarian, and endometrial cancers. Elevated HDAC expression correlated with poor prognosis and highly malignant cancer subtypes. Gene Set Enrichment Analysis revealed positive associations between HDAC expression and tumor proliferation signature, while negative associations were found with tumor inflammation signature. Increased HDAC expression was linked to reduced infiltration of natural killer (NK), NKT, and CD8+ T cells, along with negative associations with the expression of PSMB10, NKG7, CCL5, CD27, HLA-DQA1, and HLA-DQB1. In a murine 4T1 breast cancer model, treatment with suberoylanilide hydroxamic acid (SAHA; HDAC inhibitor) and PD-1 antibody significantly inhibited tumor growth and infiltration of CD3+ and CD8+ T cells. Real-time polymerase chain reaction revealed upregulated expressions of Psmb10, Nkg7, Ccl5, Cd8a, Cxcr6, and Cxcl9 genes, while Ctnnb1 and Myc genes were inhibited, indicating tumor suppression and immune microenvironment activation. Our study revealed that HDACs play tumor-promoting and immunosuppressive roles in gynecologic cancers, suggesting HDAC inhibitors as potential therapeutic agents for these cancers.

Identification of Bacterial Metabolites Modulating Breast Cancer Cell Proliferation and Epithelial-Mesenchymal Transition.

Breast cancer patients are characterized by the oncobiotic transformation of multiple microbiome communities, including the gut microbiome. Oncobiotic transformation of the gut microbiome impairs the production of antineoplastic bacterial metabolites. The goal of this study was to identify bacterial metabolites with antineoplastic properties. We constructed a 30-member bacterial metabolite library and screened the library compounds for effects on cell proliferation and epithelial-mesenchymal transition. The metabolites were applied to 4T1 murine breast cancer cells in concentrations corresponding to the reference serum concentrations. However, yric acid, glycolic acid, d-mannitol, 2,3-butanediol, and trans-ferulic acid exerted cytostatic effects, and 3-hydroxyphenylacetic acid, 4-hydroxybenzoic acid, and vanillic acid exerted hyperproliferative effects. Furthermore, 3-hydroxyphenylacetic acid, 4-hydroxybenzoic acid, 2,3-butanediol, and hydrocinnamic acid inhibited epithelial-to-mesenchymal (EMT) transition. We identified redox sets among the metabolites (d-mannitol-d-mannose, 1-butanol-butyric acid, ethylene glycol-glycolic acid-oxalic acid), wherein only one partner within the set (d-mannitol, butyric acid, glycolic acid) possessed bioactivity in our system, suggesting that changes to the local redox potential may affect the bacterial secretome. Of the nine bioactive metabolites, 2,3-butanediol was the only compound with both cytostatic and anti-EMT properties.

Evaluation of the anti-tumor effects of an anti-Human Epidermal growth factor receptor 2 (HER2) monoclonal antibody in combination with CD11b+/Gr-1+ myeloid cells depletion using a recombinant peptibody in 4T1-HER2 tumor model.

Clinical efficacy of Human Epidermal growth factor Receptor 2 (HER2) targeted strategies is limited due to impaired anti-tumor responses negatively regulated by immunosuppressive cells. We thus, investigated the inhibitory effects of an anti-HER2 monoclonal antibody (1T0 mAb) in combination with CD11b+/Gr-1+ myeloid cells depletion in 4T1-HER2 tumor model. BALB/c mice were challenged with human HER2-expressing 4T1 murine breast cancer cell line. A week post tumor challenge, each mouse received 50µg of a myeloid cells specific peptibody every other day, or 10mg/kg of 1T0 mAb two times a week, and their combination for two weeks. The treatments effect on tumor growth was measured by calculating tumor size. Also, the frequencies of CD11b+/Gr-1+ cells and T lymphocytes were measured by flow cytometry. Peptibody treated mice indicated tumor regression and 40% of the mice eradicated their primary tumors. The peptibody was capable to deplete notably splenic CD11b+/Gr-1+ cells as well as intratumoral CD11b+/Gr-1+ cells (P<0.0001) and led to an increased number of tumor infiltrating CD8+ T cells (3.3 folds) and also that of resident tumor draining lymph nodes (TDLNs) (3 folds). Combination of peptibody and 1T0 mAb resulted in enhanced expansion of tumor infiltrating CD4+and CD8+ T cells which was associated with tumor eradication in 60% of the mice. Peptibody is able to deplete CD11b+/Gr-1+ cells and increase anti-tumoral effects of the 1T0 mAb in tumor eradication. Thus, this myeloid population have critical roles in development of tumors and their depletion is associated with induction of anti-tumoral responses.

Murine Breast Cancer Radiosensitization Using Oxygen Microbubbles and Metformin: Vessels Are the Key.

Radiotherapy is a cornerstone of cancer treatment, but tumor hypoxia and resistance to radiation remain significant challenges. Vascular normalization has emerged as a strategy to improve oxygenation and enhance therapeutic outcomes. In this study, we examine the radiosensitization potential of vascular normalization using metformin, a widely used anti-diabetic drug, and oxygen microbubbles (OMBs). We investigated the synergistic action of metformin and OMBs and the impact of this therapeutic combination on the vasculature, oxygenation, invasiveness, and radiosensitivity of murine 4T1 breast cancer. We employed in vivo Doppler ultrasonographic imaging for vasculature analysis, electron paramagnetic resonance oximetry, and immunohistochemical assessment of microvessels, perfusion, and invasiveness markers. Our findings demonstrate that both two-week metformin therapy and oxygen microbubble treatment normalize abnormal cancer vasculature. The combination of metformin and OMB yielded more pronounced and sustained effects than either treatment alone. The investigated therapy protocols led to nearly twice the radiosensitivity of 4T1 tumors; however, no significant differences in radiosensitivity were observed between the various treatment groups. Despite these improvements, resistance to treatment inevitably emerged, leading to the recurrence of hypoxia and an increased incidence of metastasis.

Introduction of an Ultraviolet C-Irradiated 4T1 Murine Breast Cancer Whole-Cell Vaccine Model.

The advent of immunotherapy has revolutionized cancer treatments. However, the application of immune checkpoint inhibitors may entail severe side effects, with the risk of therapeutic resistance. The generation of chimeric antigen receptor (CAR) T-cells or CAR-NK cells requires specialized molecular laboratories, is costly, and is difficult to adapt to the rapidly growing number of cancer patients. To provide a simpler but effective immune therapy, a whole-cell tumor vaccine protocol was established based on ultraviolet C (UCV)-irradiated 4T1 triple-negative breast cancer cells. The apoptosis of tumor cells after UVC irradiation was verified using resazurin and Annexin V/propidium iodide flow cytometric assays. Protective immunity was achieved in immunized BALB/c mice, showing partial remission. Adoptive transfer of splenocytes or plasma from the mice in remission showed a protective effect in the naive BALB/c mice that received a living 4T1 tumor cell injection. 4T1-specific IgG antibodies were recorded in the plasma of the mice following immunization with the whole-cell vaccine. Interleukin-2 (IL-2) and oligonucleotide 2006 (ODN2006) adjuvants were used for the transfer of splenocytes from C57BL/6 mice into cyclophosphamide-treated BALB/c mice, resulting in prolonged survival, reduced tumor growth, and remission in 33% of the cases, without the development of the graft-versus-host disease. Our approach offers a simple, cost-effective whole-cell vaccine protocol that can be administered to immunocompetent healthy organisms. The plasma or the adoptive transfer of HLA-matching immunized donor-derived leukocytes could be used as an immune cell therapy for cancer patients.

Role of Gemini Surfactants with Variable Spacers and SiO2 Nanoparticles in ct-DNA Compaction and Applications toward In Vitro/In Vivo Gene Delivery.

Compaction of calf thymus DNA (ct-DNA) by two cationic gemini surfactants, 12-4-12 and 12-8-12, in the absence and presence of negatively charged SiO2 nanoparticles (NPs) (∼100 nm) has been explored using various techniques. 12-8-12 having a longer hydrophobic spacer induces a greater extent of ct-DNA compaction than 12-4-12, which becomes more efficient with SiO2 NPs. While 50% ct-DNA compaction in the presence of SiO2 NPs occurs at ∼77 nM of 12-8-12 and ∼130 nM of 12-4-12, but a conventional counterpart surfactant, DTAB, does it at its concentration as high as ∼7 μM. Time-resolved fluorescence anisotropy measurements show changes in the rotational dynamics of a fluorescent probe, DAPI, and helix segments in the condensed DNA. Fluorescence lifetime data and ethidium bromide exclusion assays reveal the binding sites of surfactants to ct-DNA. 12-8-12 with SiO2 NPs has shown the highest cell viability (≥90%) and least cell death in the human embryonic kidney (HEK) 293 cell lines in contrast to the cell viability of ≤80% for DTAB. These results show that 12-8-12 with SiO2 NPs has the highest time and dose-dependent cytotoxicity compared to 12-8-12 and 12-4-12 in the murine breast cancer 4T1 cell line. Fluorescence microscopy and flow cytometry are performed for in vitro cellular uptake of YOYO-1-labeled ct-DNA with surfactants and SiO2 NPs using 4T1 cells after 3 and 6 h incubations. The in vivo tumor accumulation studies are carried out using a real-time in vivo imaging system after intravenous injection of the samples into 4T1 tumor-bearing mice. 12-8-12 with SiO2 has delivered the highest amount of ct-DNA in cells and tumors in a time-dependent manner. Thus, the application of a gemini surfactant with a hydrophobic spacer and SiO2 NPs in compacting and delivering ct-DNA to the tumor is proven, warranting its further exploration in nucleic acid therapy for cancer treatment.

Evaluation of efficacy of tumor-specific nanoliposomal radiosensitizer in radiotherapy

Radiotherapy is one of the cancer treatment options in which ionizing radiation is used. While the ionizing radiation used here damages the tumor cells, it also damages the surrounding healthy cells and tissues. One of the approaches used to reduce the undesirable side effects and increase the therapeutic efficacy of radiotherapy is the use of radiosensitizers. Up to now, different radiosensitizers have been investigated for this purpose and recently, nanotechnology-based radiosensitizer research has attracted much attention. In this study, the therapeutic efficacy of tumor-targeted, nano-sized liposomal radiosensitizers containing quantum dots-photosensitizer conjugate was evaluated in vivo. It was aimed to destroy more tumor cells by using same the radiation doses routinely used in cancer treatment in radiation oncology clinics or to destroy about the same amount of tumor cells by using lower radiation doses than the routinely used clinical dose. For this purpose, nano-sized, PEG-coated, and folic acid-modified tumor-specific liposomes loaded with quantum dots (QD = CdSe/ZnS) - photosensitizer (Ce6 = Clorine-e6) conjugate was prepared and their therapeutic efficacy was evaluated in 4T1 murine breast cancer cell tumor-bearing mice. Following the single dose of 10Gy 6 MV X-ray irradiation after administration of liposomal radiosensitizer, changes in animals' weights and tumor volumes were measured and animal survival was monitored to evaluate the efficacy of the treatment. Histopathological studies were also performed to examine possible damage to the tumor and normal tissues. In control groups, only X-ray irradiation was applied or no treatment was applied to mice. The results showed that the liposomal radiosensitizer plus radiotherapy killed more cancer cells than radiotherapy alone. While tumor volumes increased in the control group, approximately a 39% reduction in tumor volumes was observed in mice treated with x-ray irradiation following administration of the liposomal radiosensitizer. The obtained results showed that nano-sized liposomal radiosensitizers could be a promising radiosensitizer to achieve better treatment efficacy in radiotherapy by applying fewer radiation doses compared to clinically used radiation doses.

Mucosal vaccination with an adenoviral vector vaccine protects against pulmonary breast cancer metastasis by robust induction of tissue-resident memory T-cells in the lung

Abstract The presence of tissue-resident memory T-cells (TRM) in solid tumors positively correlates with an improved overall prognosis and responsiveness to checkpoint inhibitor therapy. Rationally designed vaccine approaches capable of inducing antitumor TRM at cancer sites are needed to harness the full potential of localized immunity in tumor therapy. Our project aims to establish tumor antigen-specific lung TRM by mucosal vaccination with an adenoviral (Ad) vaccine to prevent and treat lung metastasis in the murine 4T1 breast cancer model. We employ Ad serotype 5 vectors encoding for 4T1 tumor-associated antigens from Mage-b and MuLV gp70 for intranasal vaccination, supplemented with an Interleukin-1β-encoding vector to specifically boost lung TRM induction. Indeed, particularly if Ad-IL-1β was co-delivered, mucosal vaccination established high numbers of tumor antigen-specific CD69 +CD103 +lung TRM as shown by phenotypic and functional analyses in combination with intravascular staining. Vaccine efficacy was evaluated in a 4T1 pseudometastasis model mimicking a prophylactic vaccination against lung metastasis in breast cancer patients. Local immunization led to a significant protection against pulmonary metastasis and improved survival compared to non-vaccinated and, notably, intramuscularly immunized mice. By using FTY720 to inhibit T-cell circulation upon tumor challenge, we confirmed that TRM are crucial contributors to the vaccine efficacy. In conclusion, induction of tumor-antigen specific lung TRM are a key point in local protection against pulmonary metastasis. Ongoing studies analyze the therapeutic use of this vaccination strategy as well as its potential in combination with immune checkpoint inhibition. Supported by grants from 'Anschubfinanzierung (ELAN) im IZKF' and 'Doktor Robert Pfleger-Stiftung'.

Inhibition of Poly(ADP-ribose) Polymerase Sensitizes [177Lu]Lu-DOTAGA.(SA.FAPi)2-Mediated Radiotherapy in Triple-Negative Breast Cancer.

Fibroblast activation protein (FAP) is highly expressed in many tumor types and constitutes a promising target for tumor-specific delivery of therapeutic radionuclides. [177Lu]Lu-DOTAGA.(SA.FAPi)2 is a novel radiopharmaceutical based on a novel bidentate inhibitor of FAP that is excreted more slowly than its monomeric counterparts. Still, the efficacy of radiotherapy is mitigated by cascades of DNA damage repair signaling in tumor cells including those via Poly(ADP-ribose) polymerase (PARP). We hereby aimed to evaluate the efficacy of [177Lu]Lu-DOTAGA.(SA.FAPi)2 in combination with a PARP inhibitor, Olaparib, in the 4T1 murine triple negative breast cancer (TNBC) model. The therapeutic efficacy was visualized using 18F-FDG and [68Ga]Ga-FAPI-04 positron emission imaging/computer tomography (PET/CT). Our results demonstrated that Olaparib suppressed BALB/3T3 fibroblasts in vitro and sensitized the efficacy of [177Lu]Lu-DOTAGA.(SA.FAPi)2 in mice bearing 4T1 tumors via enhancement of DNA damage. Treatment-associated toxicity was tolerable with only mild leukopenia. Therefore, the combination of [177Lu]Lu-DOTAGA.(SA.FAPi)2 and Olaparib is a feasible treatment against TNBC.

Abstract 2890: Investigating dynamic IDO-Kyn-Ahr pathway induced tumor immunosuppression using imaging modality to optimize therapeutic Intervention

Abstract Despite great clinical responses in several cancers to immune checkpoint blockade (ICB) intervention, there persist many mechanisms of tumor evasion. Tryptophan catabolism by enzymes Indoleamine-2,3-dioxygenase 1 (IDO1) and tryptophan-2,3-dioxygenase 2 (TDO2) into metabolite L-Kynurenine (Kyn) has been shown to drive an immunosuppressive tumor microenvironment (TME). We have shown tumors overexpressing IDO1 recruit and activate the Treg/macrophage suppressive axis and blockade of aryl hydrocarbon receptor (AHR) activation abrogates this. Preclinical models demonstrate inhibition of IDO1 delayed tumor growth and increased T cell function. However, a phase III clinical trial in melanoma patients combining IDO1 inhibition with anti-PD-1 blockade showed no improved efficacy when compared to anti-PD-1 blockade. Kyn can act as a key signaling molecule through activation of AHR. We hypothesize that AHR engagement by kyn within the TME is highly dynamic and elevated AHR engagement must coincide with intervention to maximize therapeutic response. Using an AHR biosensor, we designed a construct with a dioxin response element (DRE) promoter that is engaged upon AHR-ligand complex, translocated to the nucleus, and drives a luciferase gene with a luminescence readout. This allows us to use an image-guided approach to track AHR bioactivity in real-time. In-vitro, we were able to validate our construct using transduced murine B16 melanoma and 4T1 breast cancer models. We treated transduced cells with endogenous AHR ligands, kynurenine, and AHR agonist (FICZ), and observed a significant dose-dependent increase in AHR reporter readout. Additionally, the AHR inhibitor (CH-223191) abrogated AHR activity and subsequent luminescence below control threshold. We implanted both DRE transduced B16 (B16-DRE) and 4T1 (4T1-DRE) intradermally and intramammary fat pad respectively. Tumors were imaged and we observed total luminescence flux corresponding to increased tumor growth. After three weeks, we assessed IDO, AHR, and HIF1a expression which all converge on the IDO-Kyn-AhR pathway using a broad panel of lymphocyte and myeloid markers from tumors and spleen. We identified high expression of IDO, AHR, and HIF1a markers in myeloid cells with an increased frequency of dendritic cells and macrophages. Future experiments will focus on using our reporter construct in the context of ICB treatment and assessing AHR activity overtime as well as sampling peripheral blood to assess the magnitude of tryptophan, kyn, and other polycyclic hydrocarbons including gut microbiome-derived indoles. The characterization of the IDO-Kyn-AhR modulation after therapeutic intervention will inform the rational design of treatment regimens to abrogate immune suppression associated with AHR activity in combination with ICB treatment. Citation Format: Mamadou Alpha Bah, Inna Serganova, Sadna Budhu, Tanya Schild, Jonathan F. Khan, Kayvan R. Keshari, Jedd D. Wolchok, Taha Merghoub. Investigating dynamic IDO-Kyn-Ahr pathway induced tumor immunosuppression using imaging modality to optimize therapeutic Intervention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2890.