4T1 Model Research Articles

A Metal Chelation Therapy to Effectively Eliminate Breast Cancer and Intratumor Bacteria While Suppressing Tumor Metastasis by Copper Depletion and Zinc Ions Surge.

The intratumor microbiota results in the immunosuppressive microenvironment and facilitates tumor growth and metastasis. However, developing a synergistic therapy with antitumor, antibacterial, and antimetastatic effects faces enormous challenges. Here, we propose anmetal chelation therapy to effectively eliminate tumor and intratumor bacteria and suppress tumor metastasis. Different from traditional chelation therapy that only consumes metal elements, this therapy not only eliminates the crucial metal elements for tumor metabolism but also releases new metal ions with antitumor and antibacterial properties. Based on the high demand for copper in breast cancer, we prepare a fibrous therapeutic nanoagent (Zn-PEN) by chelatingD-Penicillamine (D-PEN) with Zn2+. Firstly, Zn-PEN achieves dual inhibition of oxidative phosphorylationand glycolysis metabolism through copper depletion and Zn2+ activated cGAS-STING pathway, thus inducing tumor cell death. Secondly, Zn-PEN has the capability to eradicate Fusobacterium nucleatumin breast cancer, thereby mitigating its immunosuppressive impact on the tumor microenvironment. Finally, Zn-PEN effectively inhibits tumor metastasis through multiple routes, including the inhibition of epithelial-mesenchymal transition process, activation of cGAS-STING pathway, and elimination with F. nucleatum. Therefore, we verify the feasibility of Zn-PEN mediated metal chelation therapy in a 4T1 model infected with F. nucleatum, providing a new therapeutic strategy for inhibiting tumor metastasis.

A Metal Chelation Therapy to Effectively Eliminate Breast Cancer and Intratumor Bacteria While Suppressing Tumor Metastasis by Copper Depletion and Zinc Ions Surge

The intratumor microbiota results in the immunosuppressive microenvironment and facilitates tumor growth and metastasis. However, developing a synergistic therapy with antitumor, antibacterial, and antimetastatic effects faces enormous challenges. Here, we propose an metal chelation therapy to effectively eliminate tumor and intratumor bacteria and suppress tumor metastasis. Different from traditional chelation therapy that only consumes metal elements, this therapy not only eliminates the crucial metal elements for tumor metabolism but also releases new metal ions with antitumor and antibacterial properties. Based on the high demand for copper in breast cancer, we prepare a fibrous therapeutic nanoagent (Zn‐PEN) by chelating D‐Penicillamine (D‐PEN) with Zn2+. Firstly, Zn‐PEN achieves dual inhibition of oxidative phosphorylation and glycolysis metabolism through copper depletion and Zn2+ activated cGAS‐STING pathway, thus inducing tumor cell death. Secondly, Zn‐PEN has the capability to eradicate Fusobacterium nucleatum in breast cancer, thereby mitigating its immunosuppressive impact on the tumor microenvironment. Finally, Zn‐PEN effectively inhibits tumor metastasis through multiple routes, including the inhibition of epithelial‐mesenchymal transition process, activation of cGAS‐STING pathway, and elimination with F. nucleatum. Therefore, we verify the feasibility of Zn‐PEN mediated metal chelation therapy in a 4T1 model infected with F. nucleatum, providing a new therapeutic strategy for inhibiting tumor metastasis.

Green Nanotechnology Through Papain Nanoparticles: Preclinical in vitro and in vivo Evaluation of Imaging Triple-Negative Breast Tumors.

Recent advancements in nanomedicine and nanotechnology have expanded the scope of multifunctional nanostructures, offering innovative solutions for targeted drug delivery and diagnostic agents in oncology and nuclear medicine. Nanoparticles, particularly those derived from natural sources, hold immense potential in overcoming biological barriers to enhance therapeutic efficacy and diagnostic accuracy. Papain, a natural plant protease derived from Carica papaya, emerges as a promising candidate for green nanotechnology-based applications due to its diverse medicinal properties, including anticancer properties. This study presents a novel approach in nanomedicine and oncology, exploring the potential of green nanotechnology by developing and evaluating technetium-99m radiolabeled papain nanoparticles (99mTc-P-NPs) for imaging breast tumors. The study aimed to investigate the efficacy and specificity of these nanoparticles in breast cancer models through preclinical in vitro and in vivo assessments. Papain nanoparticles (P-NPs) were synthesized using a radiation-driven method and underwent thorough characterization, including size, surface morphology, surface charge, and cytotoxicity assessment. Subsequently, P-NPs were radiolabeled with technetium-99m (99mTc), and in vitro and in vivo studies were conducted to evaluate cellular uptake at tumor sites, along with biodistribution, SPECT/CT imaging, autoradiography, and immunohistochemistry assays, using breast cancer models. The synthesized P-NPs exhibited a size mean diameter of 9.3 ± 1.9 nm and a spherical shape. The in vitro cytotoxic activity of native papain and P-NPs showed low cytotoxicity in HUVEC, MDA-MB231, and 4T1 cells. The achieved radiochemical yield was 94.2 ± 3.1% that were sufficiently stable (≥90%) for 6 h. The tumor uptake achieved in the 4T1 model was 2.49 ± 0.32% IA/g at 2h and 1.51 ± 0.20% IA/g at 6h. In the spontaneous breast cancer model, 1.19 ± 0.20% IA/g at 2h and 0.86 ± 0.31% IA/g at 6h. SPECT/CT imaging has shown substantial tumor uptake of the new nanoradiopharmaceutical and clear tumor visualization. 99mTc-P-NPs exhibited a high affinity to tumoral cells confirmed by ex vivo autoradiography and immunohistochemistry assays. The findings underscore the potential of green nanotechnology-driven papain nanoparticles as promising agents for molecular imaging of breast and other tumors through SPECT/CT imaging. The results represent a substantial step forward in the application of papain nanoparticles as carriers of diagnostic and therapeutic radionuclides to deliver diagnostic/therapeutic payloads site-specifically to tumor sites for the development of a new generation of nanoradiopharmaceuticals.

Flt3 ligand augments immune responses to soluble PD1-based DNA vaccine via expansion of type 1 conventional DCs

DNA vaccines are prospective for their efficient manufacturing process, but their immunogenicity is limited as they cannot efficiently induce CD8+ T cell responses. A promising approach is to induce cross-presentation by targeting antigens to DCs. Flt3L can expand the number of type 1 conventional DCs and thereby improve cross-presentation. In this study, we first constructed a DNA vaccine expressing soluble PD1 and found that the therapeutic effect of targeting DCs with only the sPD1 vaccine was limited. When combined the vaccine with Flt3L, the anti-tumor effect was significantly enhanced. Considering the complexity of tumors and that a single method may not be able to activate a large number of effective CD8+ T cells, we combined different drugs and the vaccine with Flt3L based on the characteristics of different tumors. In 4T1 model, we reduced Tregs through cyclophosphamide. In Panc02 model, we increased activated DCs by using aCD40. Both strategies triggered strong CD8+ T cell responses and significantly improved the therapeutic effect. Our study provides important support for the clinical exploration of DC-targeted DNA vaccines in combination with Flt3L.

Optical parameters of healthy and tumor breast tissues in mice.

Knowledge of the optical parameters of tumors is important for choosing the correct laser treatment parameters. In this paper, optical properties and refraction indices of breast tissue in healthy mice and a 4T1 model mimicking human breast cancer have been measured. A significant decrease in both the scattering and refractive index of tumor tissue has been observed. The change in tissue morphology has induced the change in the slope of the scattering spectrum. Thus, the light penetration depth into tumor has increased by almost 1.5-2 times in the near infrared "optical windows." Raman spectra have shown lower lipid content and higher protein content in tumor. The difference in the optical parameters of the tissues under study makes it possible to reliably differentiate them. The results may be useful for modeling the distribution of laser radiation in healthy tissues and cancers for deriving optimal irradiation conditions in photodynamic therapy.

Garlic peel extract as an antioxidant inhibits triple‐negative breast tumor growth and angiogenesis by inhibiting cyclooxygenase‐2 expression

AbstractGarlic peels are frequently disposed of as agro‐waste; their bioactivity and physiological activity for health benefits and disease protection are neglected. This study aims to examine the potential inhibitory effects of garlic peel extract as an antioxidant on 4 T1 triple‐negative breast cancer (TNBC) tumors in mice. The bioactive constituents of garlic peel were identified through HPLC‐MS/MS analysis, while the antioxidant properties of garlic peel extract were assessed using peroxyl radical scavenging capacity (PSC) and cellular antioxidant activity (CAA) assays. Subsequently, the inhibitory effects of garlic peel extract on 4T1 tumor growth were evaluated using a 4T1 model. The results showed that 433 polyphenol compounds were found in garlic peel extract; among them, flavonoids and phenolic acid are the primary polyphenols with natural antioxidant activity, and both high and low concentrations of the extract exhibited tumor‐suppressive effects. Immunohistochemistry was employed to assess the expression levels of COX‐2, CD31, VEGFA, MMP2, and MMP9 in tumor tissues in order to investigate the antioxidant properties of garlic peel extract, specifically its ability to suppress COX‐2 expression. The findings of this study offer a foundation for the advancement of garlic peel‐based functional products and contribute to the identification of potential anti‐cancer agents and therapeutic targets.

High-Lactate-Metabolizing Photosynthetic Bacteria Reprogram Tumor Immune Microenvironment.

The elevated levels of lactate in tumor tissue play a pivotal role in fostering an immunosuppressive microenvironment. Therefore, efficiently reducing lactate levels to reprogram tumor immune microenvironment (TIM) is considered a crucial step for boosted immunotherapy. Here, a high-lactate-metabolizing photosynthetic bacteria (LAB-1) is selectively screened for TIM reprogramming, which then improves the efficacy of tumor immunotherapy. The culture medium for LAB-1 screening is initially developed through an orthogonal experiment, simulating the tumor microenvironment (TME) and utilizing lactate as the sole organic carbon source. As demonstrated in a murine 4T1 model, LAB-1 colonizes the TME selectively, resulting in a significant reduction in lactate levels and a subsequent increase in pH values within the tumor tissue. Furthermore, single-cell RNA sequencing analysis reveals that LAB-1 effectively reprograms the TIM, thereby enhancing the effectiveness of antitumor immune therapy. This approach of utilizing lactate-consuming bacteria represents a potent tool for augmenting tumor immunotherapy efficiency.

Photosynthetic bacteria-based whole-cell inorganic-biohybrid system for multimodal enhanced tumor radiotherapy

The whole-cell inorganic-biohybrid systems show special functions and wide potential in biomedical application owing to the exceptional interactions between microbes and inorganic materials. However, the hybrid systems are still in stage of proof of concept. Here, we report a whole-cell inorganic-biohybrid system composed of Spirulina platensis and gold nanoclusters (SP-Au), which can enhance the cancer radiotherapy through multiple pathways, including cascade photocatalysis. Such systems can first produce oxygen under light irradiation, then convert some of the oxygen to superoxide anion (•O2−), and further oxidize the glutathione (GSH) in tumor cells. With the combination of hypoxic regulation, •O2− production, GSH oxidation, and the radiotherapy sensitization of gold nanoclusters, the final radiation is effectively enhanced, which show the best antitumor efficacy than other groups in both 4T1 and A549 tumor models. Moreover, in vivo distribution experiments show that the SP-Au can accumulate in the tumor and be rapidly metabolized through biodegradation, further indicating its application potential as a new multiway enhanced radiotherapy sensitizer.

Evaluating the immunologically “cold” tumor microenvironment after treatment with immune checkpoint inhibitors utilizing PET imaging of CD4 + and CD8 + T cells in breast cancer mouse models

BackgroundImmune-positron emission tomography (PET) imaging with tracers that target CD8 and granzyme B has shown promise in predicting the therapeutic response following immune checkpoint blockade (ICB) in immunologically “hot” tumors. However, immune dynamics in the low T-cell infiltrating “cold” tumor immune microenvironment during ICB remain poorly understood. This study uses molecular imaging to evaluate changes in CD4 + T cells and CD8 + T cells during ICB in breast cancer models and examines biomarkers of response.Methods[89Zr]Zr-DFO-CD4 and [89Zr]Zr-DFO-CD8 radiotracers were used to quantify changes in intratumoral and splenic CD4 T cells and CD8 T cells in response to ICB treatment in 4T1 and MMTV-HER2 mouse models, which represent immunologically “cold” tumors. A correlation between PET quantification metrics and long-term anti-tumor response was observed. Further biological validation was obtained by autoradiography and immunofluorescence.ResultsFollowing ICB treatment, an increase in the CD8-specific PET signal was observed within 6 days, and an increase in the CD4-specific PET signal was observed within 2 days in tumors that eventually responded to immunotherapy, while no significant differences in CD4 or CD8 were found at the baseline of treatment that differentiated responders from nonresponders. Furthermore, mice whose tumors responded to ICB had a lower CD8 PET signal in the spleen and a higher CD4 PET signal in the spleen compared to non-responders. Intratumoral spatial heterogeneity of the CD8 and CD4-specific PET signals was lower in responders compared to non-responders. Finally, PET imaging, autoradiography, and immunofluorescence signals were correlated when comparing in vivo imaging to ex vivo validations.ConclusionsCD4- and CD8-specific immuno-PET imaging can be used to characterize the in vivo distribution of CD4 + and CD8 + T cells in response to immune checkpoint blockade. Imaging metrics that describe the overall levels and distribution of CD8 + T cells and CD4 + T cells can provide insight into immunological alterations, predict biomarkers of response to immunotherapy, and guide clinical decision-making in those tumors where the kinetics of the response differ.

Magnesium microspheres to enhance lipiodol-mediated transarterial chemo-embolization (TACE) of hepatocellular carcinoma: From bench to a pilot clinical study.

e16204 Background: Transarterial chemoembolization (TACE) has been extensively used in clinic to treat unresectable hepatocellular carcinoma (HCC). However, arterial embolization would worsen the hostile physicochemical features in the tumor microenvironment (TME) by further reducing its pH and increasing hypoxia, leading to immunosuppressive TME and drug resistance. Methods: Magnesium microspheres (Mg MSs), acting as H2 donors and pH modulators, were prepared via gas atomization pulverization. Characterization included inverted optical microscopy, SEM, and XRD analysis. Their H2 generation and acid neutralization capabilities were confirmed. The Lip-Mg dispersion (Mg MSs dispersed in lipiodol) was tested for therapeutic efficacy in H22 murine liver tumor-bearing mice, 4T1, and CT26 subcutaneous tumor models, as well as in vivo interventional TAE therapy for rabbit orthotopic liver tumors. The modulated capacities of Lip-Mg in H2 gas generation, acid neutralization, and promotion of antitumor immune response were investigated in vivo. Finally, a single-center, single-arm, open-label trial (ChiCTR2200064999) evaluated the safety and efficacy of TACE treatment with Lip-Mg (M-TACE) in Chinese HCC patients. Results: Inverted optical microscopy and SEM images confirmed uniform, smooth Mg microspheres (~60 μm) with a grayish-white color. In acidic buffer (pH 6.4), Mg microspheres rapidly increased pH, as measured by pH meter or fluorescent probe. Lip-Mg treatment significantly inhibited tumor growth compared to controls, prolonging mouse survival by ~2-fold, validated in 4T1 and CT26 models. Ultrasonic and multispectral fluorescence imaging demonstrated Lip-Mg's ability to release H2 and neutralize pH in vivo, reversing the immunosuppressive tumor microenvironment. Clinical study showed manageable treatment-related adverse events, with no unexpected toxicities and negligible impact on serum magnesium levels, confirming the safety of Mg microspheres for M-TACE. According to mRECIST, M-TACE exhibited high CR (61.5%), PR (38.5%), and ORR (100%) rates, with an overall response rate and CR rate of 90% and 60%, respectively. Conclusions: Metallic Mg microspheres (~60 μm) were developed to enhance lipiodol-mediated TAE/TACE for HCC. When combined with lipiodol and injected, they neutralized the acidic TME, triggered hydrogen therapy, and reversed immunosuppression, effectively inhibiting tumor growth. Lip-Mg showed superior outcomes to lipiodol alone for TAE therapy, with M-TACE treatment exhibiting higher ORR and CR rates than cTACE. Post-M-TACE, adverse events were managed well without treatment discontinuations. Our study introduces Mg microspheres as a promising embolic device for M-TACE therapy, potentially improving TACE benefits. Clinical trial information: ChiCTR2100050327.

Combining the potency of a neoantigen-expressing DNA vector, an anti-CTLA-4 antibody, and an attenuated poxvirus in a personalized immunization platform: Preclinical studies of ODI-2001.

e14674 Background: Immune checkpoint inhibitors (ICI) have transformed cancer treatment. Their impressive effectiveness is crucially dependent on pre-existing neoepitope-specific immune responses. ODI-2001 aims at increasing the immune response against tumors by combining a DNA expressing multiple neoepitopes of the tumor, a MVA vaccinia virus as physiologic adjuvant and DNA carrier, and an anti-CTLA4 antibody as amplifier of the immune response. Methods: ODI-2001 was administered by repeated subcutaneous injections after tumor cells implantation in mice melanoma model B16F10 and colorectal cancer model CT26, and both before and after implantation in breast cancer model 4T1. In B16F10 model combination of ODI-2001 with anti-PD1 was assessed also. Neoepitopes were chosen from literature in B16F10 and CT26 models. In 4T1 model they were predicted using a dedicated neoepitope prediction algorithm (ImmunoEngine, myNEO, Gent, Belgium). Tumor volume and mice survival were assessed over time. Tumors were also analysed by immunofluorescence for immune cell infiltration. Each experiment received approval from the ethics committee for animals. Results: A significant improvement in survival was observed in mice treated with ODI-2001 in B16F10, and CT26 syngeneic mice models. In B16F10 the combination of ODI-2001 with anti-PD1 antibody demonstrated synergistic activity and a significant improvement in survival, while anti-PD1 alone or combined with anti-CTLA-4 failed to show any therapeutic effect (ODI-2001 + anti-PD1 vs. anti-CTLA-4 + anti-PD1 p<0.01). ODI-2001 treatment was associated with tumor rejections in the three animal models. In 4T1 model the ImmunoEngine epitope prediction algorithm demonstrated an improved efficacy as compared to a set of neoepitopes chosen from the literature. In B16F10, following immunization with ODI-2001, an increased CD8 immune cell infiltration of the tumor was documented. ODI-2001 was well tolerated by the treated animals. Conclusions: These data demonstrate a significant antitumoral activity of ODI-2001 as a neoantigen immunization platform across different preclinical cancer models, both in monotherapy and combined with anti-PD1. ODI-2001 represents a promising option to induce de novo cancer-specific immune responses, potentially increasing the proportion of patients responding to immunotherapy treatments. Based on this preclinical program a phase I with ODI-2001 in patients with advanced colorectal carcinoma is currently under preparation.

Engineered Immunologic Niche Monitors Checkpoint Blockade Response and Probes Mechanisms of Resistance.

Antibodies to programmed cell death protein1 (anti-PD-1) have become a promising immunotherapy for triple negative breast cancer (TNBC), blocking PD-L1 signaling from pro-tumor cells through T cell PD-1 receptor binding. Nevertheless, only 10-20% of PD-L1+ metastatic TNBC patients who meet criteria benefit from ICB, and biomarkers to predict patient response have been elusive. We have previously developed an immunological niche, consisting of a microporous implant in the subcutaneous space, that supports tissue formation whose immune composition is consistent with that within vital organs. Herein, we investigated dynamic gene expression within this immunological niche to provide biomarkers of response to anti-PD-1. In a 4T1 model of metastatic TNBC, we observed sensitivity and resistance to anti-PD-1 based on primary tumor growth and survival. The niche was biopsied before, during, and after anti-PD-1 therapy, and analyzed for cell types and gene expression indicative of treatment refractivity. Myeloid cell-to-lymphocyte ratios were altered between ICB-sensitivity and resistance. Longitudinal analysis of gene expression implicated dynamic myeloid cell function that stratified sensitivity from resistance. A niche-derived gene signature predicted sensitivity or resistance prior to therapy. Analysis of the niche to monitor immunotherapy response presents a new opportunity to personalize care and investigate mechanisms underlying treatment resistance.

Prostate Specific Membrane Antigen Expression in a Syngeneic Breast Cancer Mouse Model

PurposeProstate specific membrane antigen (PSMA) has been studied in human breast cancer (BCa) biopsies, however, lack of data on PSMA expression in mouse models impedes development of PSMA-targeted therapies, particularly in improving breast conserving surgery (BCS) margins. This study aimed to validate and characterize the expression of PSMA in murine BCa models, demonstrating that PSMA can be utilized to improve therapies and imaging techniques.MethodsMurine triple negative breast cancer 4T1 cells, and human cell lines, MDA-MB-231, MDA-MB-468, implanted into the mammary fat pads of BALB/c mice, were imaged by our PSMA targeted theranostic agent, PSMA-1-Pc413, and tumor to background ratios (TBR) were calculated to validate selective uptake. Immunohistochemistry was used to correlate PSMA expression in relation to CD31, an endothelial cell biomarker highlighting neovasculature. PSMA expression was also quantified by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR).ResultsAccumulation of PSMA-1-Pc413 was observed in 4T1 primary tumors and associated metastases. Average TBR of 4T1 tumors were calculated to be greater than 1.5—ratio at which tumor tissues can be distinguished from normal structures—at peak accumulation with the signal intensity in 4T1 tumors comparable to that in high PSMA expressing PC3-pip tumors. Extraction of 4T1 tumors and lung metastases followed by RT-PCR analysis and PSMA-CD31 co-staining shows that PSMA is consistently localized on tumor neovasculature with no expression in tumor cells and surrounding normal tissues.ConclusionThe selective uptake of PSMA-1-Pc413 in these cancer tissues as well as the characterization and validation of PSMA expression on neovasculature in this syngeneic 4T1 model emphasizes their potential for advancements in targeted therapies and imaging techniques for BCa. PSMA holds great promise as an oncogenic target for BCa and its associated metastases.

Multifactoral immune modulation potentiates durable remission in multiple models of aggressive malignancy.

Tumors typically lack canonical danger signals required to activate adaptive immunity and also frequently employ substantial immunomodulatory mechanisms that downregulate adaptive responses and contribute to escape from immune surveillance. Given the variety of mechanisms involved in shielding tumors from immune recognition, it is not surprising that single-agent immunomodulatory approaches have been largely unsuccessful in generating durable antitumor responses. Here we report a unique combination of immunomodulatory and cytostatic agents that recondition the tumor microenvironment and eliminate complex and/or poor-prognosis tumor types including the non-immunogenic 4T-1 model of TNBC, the aggressive MOC-2 model of HNSCC, and the high-risk MYCN-amplified model of neuroblastoma. A course of therapy optimized for TNBC cured a majority of tumors in both ectopic and orthotopic settings and eliminated metastatic spread in all animals tested at the highest doses. Immune responses were transferable between therapeutic donor and naïve recipient through adoptive transfer, and a sizeable abscopal effect on distant, untreated lesions could be demonstrated experimentally. Similar results were observed in HNSCC and neuroblastoma models, with characteristic remodeling of the tumor microenvironment documented in all model systems. scRNA-seq analysis implicated upregulation of innate immune responses and antigen presentation in tumor cells and the myeloid cell compartment as critical early events. This analysis also highlighted the potential importance of the autonomic nervous system in the governance of inflammatory processes. The data indicate that the targeting of multiple pathways and mechanisms of action can result in substantial synergistic antitumor effects and suggest follow-up in the neoadjuvant setting may be warranted.

Abstract PO2-24-10: Anti-Tumorigenic Immuno-Microenvironment by AB-329, a Potent and Selective AXL inhibitor, combined with paclitaxel in Triple-Negative Breast Cancer Preclinical Model

Abstract BACKGROUND The Gas6/AXL signaling pathway plays a substantial role in cancer cell survival, proliferation, immunosuppression, and metastasis. Elevated AXL expression is associated with advanced cancer stages and decreased overall survival rates, underscoring its potential as a therapeutic target for metastatic cancers. We investigated this possibility by utilizing AB-329, a selective small molecule AXL kinase inhibitor, to treat triple-negative breast cancer (TNBC). TNBC, typified by the lack of ER, PR, and HER2 expression, frequently displays mesenchymal characteristics and carries a significant risk of metastasis and an immunosuppressive tumor microenvironment. METHODS We assessed AXL protein expression in 28 human and 11 mouse breast cancer cell lines using Western blotting. The Microarray database was utilized to examine AXL gene expression in 40 human breast cancer cells. The Sulforhodamine B proliferation and soft agar assays measured the antiproliferative effect of AB-329 in TNBC cell lines. The in vitro anti-metastatic impact of AB-329 was evaluated through migration and invasion assays. We appraised the anti-tumor effects of AB-329 in combination with paclitaxel using 4T1 and E0771-LMB mouse TNBC xenograft models. We used FACS and CIBERSORTx to perform immune cell type profiling in E0771-LMB xenograft tumor tissues. To understand AB-329’s immunomodulatory effects in tumors, single-cell RNA sequencing was conducted in a humanized mouse model with SUM149 human TNBC xenografts. RESULTS Microarray data revealed higher AXL expression in TNBC cells compared to non-TNBC cells. Western blotting supported this, showing higher AXL protein expression, particularly in basal and mesenchymal subtypes of TNBC cells, compared to luminal breast cancer cells. AB-329 presented moderate antiproliferative effects as a monotherapy in the TNBC cell lines tested, with half-maximal inhibitory concentrations exceeding 5 µM. Nevertheless, combining AB-329 with paclitaxel resulted in significant growth inhibition in all tested TNBC cell lines (p < 0.05). Importantly, AB-329 alone could inhibit TNBC migration and invasion at a concentration of 1 µM (p < 0.01) and showed enhanced inhibition rates when paired with paclitaxel (p < 0.05). The combination of AB-329 and paclitaxel considerably reduced tumor growth in the 4T1 and E0771-LMB xenograft models compared to paclitaxel or AB-329. Total RNA sequencing and CIBERSORTx analysis indicated that the AB-329 and paclitaxel combination increased Natural Killer (NK) cells’ infiltration into E0771-LMB mouse TNBC xenograft tumors compared to single agent-treated tumors. A nearly 2.5-fold increase in NKT cell infiltration was also detected with AB-329 treatment compared to Control in SUM149 human TNBC xenografts. CONCLUSIONS Our preclinical data suggest that AB-329, an AXL inhibitor, could synergize effectively with paclitaxel in TNBC treatment, notably inhibiting tumor cell proliferation and migration while enhancing NK cell infiltration. Intriguingly, we observed an increased infiltration of NKT cells into tumors following AB-329 treatment, which may promote an anti-tumorigenic immuno-microenvironment. These results underscore the necessity for further investigation into the role of AB-329 in modulating the tumor immuno-microenvironment through AXL pathway inhibition and accentuate the potential for combining AB-329 with immunotherapy. Citation Format: Dileep Reddy Rampa, Jon Fuson, Huey Liu, Max Pan, Naoto Ueno, Jangsoon Lee. Anti-Tumorigenic Immuno-Microenvironment by AB-329, a Potent and Selective AXL inhibitor, combined with paclitaxel in Triple-Negative Breast Cancer Preclinical Model [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 PO2-24-10.

Abstract PO1-18-04: Neoadjuvant pembrolizumab + decitabine followed by standard neoadjuvant chemotherapy for locally advanced HER2- breast cancer (NCT02957968)

Abstract Background: Higher levels of stromal tumor-infiltrating lymphocytes (sTILs) in breast cancers are associated with increased likelihood of pathologic complete response (pCR) to chemotherapy and improved outcomes. DNA methyltransferase inhibitors (DNMTi) can augment immune responses to cancers by upregulating tumor antigen and MHC expression, decreasing numbers and activity of myeloid derived suppressor cells (MDSC), and increasing responsiveness of T lymphocytes. We have shown that the DNMTi decitabine augments the effectiveness of immunotherapy against murine triple negative breast cancer (TNBC) using murine 4T1 and E0771 mammary carcinoma models. Methods: In a single-arm phase 2 study, patients with HER2-negative breast cancer who were candidates for neoadjuvant chemotherapy (NCT) received decitabine (15 mg/m2 × 4 doses over 5 days) followed by 2 doses of pembrolizumab (pembro) (200 mg, 2 weeks apart) – collectively, window immunotherapy – prior to starting NCT. Two research biopsies were obtained: 1 prior to the window immunotherapy and 1 afterwards, prior to starting NCT. Biopsies were analyzed using established procedures to quantify sTILs and PD-L1 expression. Patients proceeded to NCT and tumor resection per standard of care and were followed up for immune-related adverse events (irAEs) and response. The primary endpoint was change in sTILs. Key secondary endpoints were occurrence of irAEs and pCR following neoadjuvant treatment. After the study was opened, reported results of a phase 3 trial (KEYNOTE 522) led to allowing patients with TNBC to receive additional pembro (200 mg q3w) concurrently with standard NCT and adjuvantly following resection (Cohort A2). Results: 46 patients (median age 54.5 yrs, range 28-72; 71.7% White, 28.3% Black; 100% female) were enrolled and treated on study. 21 patients had TNBC and did not receive neoadjuvant pembro concurrently with NCT nor adjuvant pembro (Cohort A), 7 patients had TNBC and did receive neoadjuvant and/or adjuvant pembro (Cohort A2), and 18 patients were ER+ or PR+ and received neither concurrent nor adjuvant pembro (Cohort B). Blood samples collected after decitabine administration before the first pembro dose showed a 59% decrease (P< 0.01) in monocytic MDSCs compared to baseline; decrease in granulocytic MDSCs was not statistically significant. 37 patients had paired biopsies adequate for sTIL evaluation with a mean change from 23.4% to 30.3% (absolute change 6.9%, P< 0.001). Cohorts A/A2 experienced an absolute sTIL increase of 7.4% (P< 0.01); Cohort B experienced absolute sTIL increase of 6.1% (P=0.01). PD-L1 expression in tumors (H-score using MoAb 22C3 clone) increased by 43% (P< 0.01) across all cohorts. 16 of the 39 patients (41.0%) who proceeded to resection achieved pCR (n=12 of 27 [44.4%] in Cohorts A/A2 and n=4 of 13 [30.8%] in Cohort B). The most frequently reported irAEs were adrenal insufficiency (n=5, 10.9%), maculopapular rash (n=3, 6.5%), and hypothyroidism (n=3, 6.5%). Conclusions: Treatment in the pre-neoadjuvant window with decitabine and pembro could potentially sensitize breast cancers to standard NCT by recruitment of TILs to the tumor tissue. The treatment was well -tolerated at the tested doses. Funding: financial support and drug (pembrolizumab) were provided by Merck Citation Format: Harry Bear, Xiaoyan Deng, Dipankar Bandyopadhyay, Michael Idowu, Maciej Kmieciak, Monique Williams, Giovanni Archer, Lindsey Gwaltney, Patrick Dillon, Daniel Flora, Daniel Stover, Andrew Poklepovic, Mary Hackney, Masey Ross, Hetal Vachhani, Raphael Louie, Kandace McGuire, Amelia Grover, Tasnim Rahman, Amber Hendrix. Neoadjuvant pembrolizumab + decitabine followed by standard neoadjuvant chemotherapy for locally advanced HER2- breast cancer (NCT02957968) [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 PO1-18-04.

Turning anecdotal irradiation-induced anticancer immune responses into reproducible in situ cancer vaccines via disulfiram/copper-mediated enhanced immunogenic cell death of breast cancer cells

Irradiation (IR) induces immunogenic cell death (ICD) in tumors, but it rarely leads to the abscopal effect (AE); even combining IR with immune checkpoint inhibitors has shown only anecdotal success in inducing AEs. In this study, we aimed to enhance the IR-induced immune response and generate reproducible AEs using the anti-alcoholism drug, disulfiram (DSF), complexed with copper (DSF/Cu) to induce tumor ICD. We measured ICD in vitro and in vivo. In mouse tumor models, DSF/Cu was injected intratumorally followed by localized tumor IR, creating an in situ cancer vaccine. We determined the anticancer response by primary tumor rejection and assessed systemic immune responses by tumor rechallenge and the occurrence of AEs relative to spontaneous lung metastasis. In addition, we analyzed immune cell subsets and quantified proinflammatory and immunosuppressive chemokines/cytokines in the tumor microenvironment (TME) and blood of the vaccinated mice. Immune cell depletion was investigated for its effects on the vaccine-induced anticancer response. The results showed that DSF/Cu and IR induced more potent ICD under hypoxia than normoxia in vitro. Low-dose intratumoral (i.t.) injection of DSF/Cu and IR(12Gy) demonstrated strong anti-primary and -rechallenged tumor effects and robust AEs in mouse models. These vaccinations also increased CD8+ and CD4+ cell numbers while decreasing Tregs and myeloid-derived suppressor cells in the 4T1 model, and increased CD8+, dendritic cells (DC), and decreased Treg cell numbers in the MCa-M3C model. Depleting both CD8+ and CD4+ cells abolished the vaccine’s anticancer response. Moreover, vaccinated tumor-bearing mice exhibited increased TNFα levels and reduced levels of immunosuppressive chemokines/cytokines. In conclusion, our novel approach generated an anticancer immune response that results in a lack of or low tumor incidence post-rechallenge and robust AEs, i.e., absence of or decreased spontaneous lung metastasis in tumor-bearing mice. This approach is readily translatable to clinical settings and may increase IR-induced AEs in cancer patients.

Polymer Backpack-Loaded Tissue Infiltrating Monocytes for Treating Cancer.

Adoptive cell therapies are dramatically altering the treatment landscape of cancer. However, treatment of solid tumors remains a major unmet need, in part due to limited adoptive cell infiltration into the tumor and in part due to the immunosuppressive tumor microenvironment. The heterogeneity of tumors and presence of nonresponders also call for development of antigen-independent therapeutic approaches. Myeloid cells offer such an opportunity, given their large presence in the immunosuppressive tumor microenvironment, such as in triple negative breast cancer. However, their therapeutic utility is hindered by their phenotypic plasticity. Here, the impressive trafficking ability of adoptively transferred monocytes is leveraged into the immunosuppressive 4T1 tumor to develop an antitumor therapy. To control monocyte differentiation in the tumor microenvironment, surface-adherent "backpacks" stably modified with interferon gamma (IFNγ) are developed to stimulate macrophage plasticity into a pro-inflammatory, antitumor phenotype, a strategy as referred to as Ornate Polymer backpacks on Tissue Infiltrating Monocytes (OPTIMs). Treatment with OPTIMs substantially reduces tumor burden in a mouse 4T1 model and significantly increases survival. Cytokine and immune cell profiling reveal that OPTIMs remodeled the tumor microenvironment into a pro-inflammatory state.

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

Abstract Background. While multiple studies have demonstrated improved clinical outcomes in patients receiving concurrent chemotherapy and immunotherapy, clinical synergy may be limited by the destructive effects of DNA-damaging chemotherapy on non-cancer cells, including key constituents of anti-tumor immunity (e.g. mature T cells). Furthermore, when first-line chemotherapy fails, subsequent lines of chemotherapy produce lower response rates. Thus, new approaches that eradicate cancer cells while preserving immune cells may be required to more efficiently induce anti-tumor immunity as monotherapy or in combination with immunotherapy. Neutrophil elastase (ELANE), a neutrophil-derived serine protease, kills a wide range of cancer cells without harming immune cells through a unique mechanism involving histone H1 and the death domain of CD95. Here, we compared N17350, an optimized biologic leveraging the ELANE pathway, and chemotherapies for their ability to kill cancer cells and preserve immune cells from primary tumors of ovarian cancer (OvCa) patients and evaluated efficacy in OvCa patient cell-derived xenografts (CDXs) and a 4T1 model of metastatic breast cancer. Methods. For in vitro studies, cancer cells and CD45+ immune cells were isolated from primary tumors of chemotherapy naïve and experienced OvCa patients. Cells were treated with N17350, oxaliplatin, or doxorubicin 24h post-isolation, and viability was quantified by calcein-AM. For in vivo studies, primary OvCa cells were injected into nude mice to create CDX models. Mice were treated with 2 doses of N17350 (400μg/100mm3, intratumoral) or carboplatin (100mg/kg, intraperitoneal), and effects on tumor growth were quantified. In 4T1 model, N17350 was compared to oxaliplatin, alone or in combination with anti-CTLA4, and effects on tumor growth and immune profile were assessed. Results. N17350 killed primary cancer cells from all OvCa patients tested but was well tolerated by CD45+ tumor immune cells from the same patients. In contrast, doxorubicin and oxaliplatin showed similar toxicity to both cell types. N17350 killed cancer cells from chemotherapy naïve and experienced patients with equal efficacy, while doxorubicin and oxaliplatin showed less efficacy in patients previously treated with chemotherapy. N17350 rapidly regressed tumors in all OvCa CDX models and exhibited markedly improved efficacy over carboplatin. In the 4T1 model, N17350 outperformed oxaliplatin in terms of regressing tumors, elevating tumor immune cells, and combining with anti-CTLA4. Conclusions. Our data demonstrate that N17350 kills OvCa tumors in vitro and in vivo while sparing CD45+ immune cells in both chemotherapy naïve and experienced patients. Together with previous data showing that N17350 induces immunogenic tumor cell death, these findings suggest that N17350 could improve clinical outcomes compared to chemoimmunotherapy. Citation Format: Chang Cui, Ravindra Gujar, Christine Lee, Maria Fumagalli, Nicole Martinez, Hannah Liu, Nicole Grigaitis, Sonia Feau, Afshin Bahador, Alain P. Algazi, Lev Becker. N17350 kills cancer cells, spares immune cells, and regresses CDX tumors from chemotherapy naive and experienced patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5895.

Abstract 1187: Sensitization to chemo-immunotherapy by targeting TGF-β in preclinical triple negative breast cancer models

Abstract Metastatic triple-negative breast cancer (mTNBC) is refractory to anti-PD-(L)1 immunotherapy. In order to sensitize it, preclinical data show the value of adding chemotherapy treatment for its ability to induce immunogenic cancer cell death (Kroemer et al, 2013). This would enable the recruitment of cytotoxic T-CD8+ lymphocytes (CTL) and thus initiate an antitumor immune response to sensitize tumors to immunotherapy. Nevertheless, the results of clinical trials using chemo-immunotherapy combinations are divergent (IMpassion 130 and 131). The IMpassion 130 trial demonstrated the efficacy of nab-paclitaxel and atezolizumab (anti-PD-L1) in PD-L1+ patients, but unfortunately, IMpassion 131 with paclitaxel did not replicate these results. The choice of chemotherapy to combine with immunotherapy appears to be crucial for inducing an antitumor immune response. Optimizing chemo-immunotherapy combinations is a major challenge in the management of TNBC. The initial aim of this project was to evaluate the immune effects of an experimental chemotherapy doublet, based on two drugs used in mTNBC: cisplatin (CDDP) and eribulin. The idea was to find an alternative to the use of taxanes in combination with anti-PD-(L)1.The effect of the two molecules, alone or in combination, was evaluated on immunogenic death stigmata, modulation of effector or immunosuppressive immune populations and stromal populations, and sensitization to anti-PD-L1 immunotherapy in the immunologically "cold" model: 4T1. The in vivo studies has been realized by generation of orthotopic 4T1 tumors, the analysis of the immune infiltration was performed by RT-qPCR, IHC and flow cytometry. In vitro, our results indicate that CDDP, and more intensely in combination with eribulin, significantly induces the various immunogenic death stigmas. In vivo, the therapeutic effect of chemotherapies is limited, with a superior effect of CDDP and the combination. Nevertheless, CTL recruitment and functionality induced by CDDP are significantly enhanced by eribulin. Despite these data pointing to a potential therapeutic synergy with anti PD-L1 immunotherapy, chemo-immunotherapy treatment fails to induce a superior treatment effect. The significant increase in a TGF-β signature induced by the combination in parallel with CTL recruitment explains this resistance. The results show that inhibition of TGF-β, combined with chemo-immunotherapy in the 4T1 model, leads to a reduction in tumor immunosuppression and intratumoral fibrosis, as well as an increase in CTL infiltration and cytotoxicity, enabling tumor sensitization. Overall, this project highlights the benefits of using an experimental doublet of CDDP and eribulin-based chemotherapies. This project demonstrates that management of chemo-induced immunosuppression mechanisms is necessary to overcome tumor resistance to immunotherapy. Citation Format: Laura Kalfeist, Fanny Ledys, Stacy Petit, Samia Kada-Mohammed, Mickael Rialland, François Ghiringhelli, Emeric Limagne, Sylvain Ladoire. Sensitization to chemo-immunotherapy by targeting TGF-β in preclinical triple negative breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1187.