Orthotopic Breast Cancer Model Research Articles

Resolution-dependent MRI-to-CT translation for orthotopic breast cancer models using deep learning

Objective.This study aims to investigate the feasibility of utilizing generative adversarial networks (GANs) to synthesize high-fidelity computed tomography (CT) images from lower-resolution MR images. The goal is to reduce patient exposure to ionizing radiation while maintaining treatment accuracy and accelerating MR image acquisition. The primary focus is to determine the extent to which low-resolution MR images can be utilized to generate high-quality CT images through a systematic study of spatial resolution-dependent magnetic resonance imaging (MRI)-to-CT image conversion.Approach.Paired MRI-CT images were acquired from healthy control and tumor models, generated by injecting MDA-MB-231 and 4T1 tumor cells into the mammary fat pad of nude and BALB/c mice to ensure model diversification. To explore various MRI resolutions, we downscaled the highest-resolution MR image into three lower resolutions. Using a customized U-Net model, we automated region of interest masking for both MRI and CT modalities with precise alignment, achieved through three-dimensional affine paired MRI-CT registrations. Then our customized models, Nested U-Net GAN and Attention U-Net GAN, were employed to translate low-resolution MR images into high-resolution CT images, followed by evaluation with separate testing datasets.Main Results.Our approach successfully generated high-quality CT images (0.142mm2) from both lower-resolution (0.282mm2) and higher-resolution (0.142mm2) MR images, with no statistically significant differences between them, effectively doubling the speed of MR image acquisition. Our customized GANs successfully preserved anatomical details, addressing the typical loss issue seen in other MRI-CT translation techniques across all resolutions of MR image inputs.Significance.This study demonstrates the potential of using low-resolution MR images to generate high-quality CT images, thereby reducing radiation exposure and expediting MRI acquisition while maintaining accuracy for radiotherapy.

Discovery of the First-in-Class Dual-Target ROCK/HDAC Inhibitor with Potent Antitumor Efficacy in Vivo That Trigger Antitumor Immunity.

Triple-negative breast cancer (TNBC) represents a highly aggressive and heterogeneous malignancy. Currently, multitarget drug approaches present a promising therapeutic approach for TNBC. Utilizing a combinatorial chemistry strategy to construct a virtual screening database, dual ROCK/HDAC-targeting benzothiophene compounds were identified. Notably, compound 10h effectively inhibits ROCK1/2 and HDAC1/2/3/6/8 while demonstrating potent antiproliferative activity against breast cancer cells. In an orthotopic mouse model of breast cancer, 10h significantly suppressed tumor growth without apparent toxicity. Importantly, 10h induced immunogenic cell death (ICD), promoted dendritic cells (DCs) maturation, and activated T cells, thereby initiating antitumor immunity. In conclusion, compound 10h is a novel dual-target ROCK/HDAC inhibitor that represents a promising treatment strategy for TNBC.

Synergistic Chemo-Immunotherapy Using pH-Responsive Nanoparticles in Breast Cancer Treatment: In Vitro and In Vivo Studies.

Recent research underscores the pivotal role of the heterogeneous multicellular interactome within the tumor microenvironment (TME) in tumor progression and survival. Tumor-associated macrophages (TAMs), among other nonmalignant cells in the TME, promote an immunosuppressive environment, fostering tumor cell survival, proliferation, and resistance. Hence, combining chemotherapy with immunomodulatory agents to transition TAMs to an immunostimulatory phenotype holds immense therapeutic potential. The present study focuses on developing tumor-responsive nanoparticles (NPs) for combined chemo-immunotherapy using resiquimod (RSQ), a TLR 7/8 agonist as an immunomodulator, and paclitaxel (PTX) as chemotherapeutics. A pH-responsive NP known as PHNP, tailored with a star-shaped PLGA conjugated with poly histidine, was engineered to selectively deliver a consistent ratio of PTX and RSQ directly to the tumor site. In vitro studies demonstrate enhanced drug release at pH 6.4, increased penetration in tumor spheroids, and increased cytotoxic efficacy against breast cancer cells. Furthermore, PHNPs activate macrophages for antitumor activity. In vivo studies demonstrated a notable rise in plasma AUC and improved delivery of drugs to the tumor using PHNPs, resulting in enhanced effectiveness against tumor growth in a mouse orthotopic breast cancer model. Notably, PHNP treatment elevated intratumoral ROS and apoptosis levels and inhibited lung metastasis. Overall, this study underscores the potential of the PTX and RSQ combination as a prospective combined chemo-immunotherapeutic modality.

Inhibition of STAT3 by 2-Methoxyestradiol suppresses M2 polarization and protumoral functions of macrophages in breast cancer

BackgroundBreast cancer metastasis remains the leading cause of cancer-related deaths in women worldwide. Infiltration of tumor-associated macrophages (TAMs) in the tumor stroma is known to be correlated with reduced overall survival. The inhibitors of TAMs are sought after for reprogramming the tumor microenvironment. Signal transducer and activator of transcription 3 (STAT3) is well known to contribute in pro-tumoral properties of TAMs. 2-Methoxyestradiol (2ME2), a potent anticancer and antiangiogenic agent, has been in clinical trials for treatment of breast cancer. Here, we investigated the potential of 2ME2 in modulating the pro-tumoral effects of TAMs in breast cancer.MethodsTHP-1-derived macrophages were polarized to macrophages with or without 2ME2. The effect of 2ME2 on macrophage surface markers and anti-inflammatory genes was determined by Western blotting, flow cytometry, immunofluorescence, qRT‒PCR. The concentration of cytokines secreted by cells was monitored by ELISA. The effect of M2 macrophages on malignant properties of breast cancer cells was determined using colony formation, wound healing, transwell, and gelatin zymography assays. An orthotopic model of breast cancer was used to determine the effect of 2ME2 on macrophage polarization and metastasis in vivo.ResultsFirst, our study found that polarization of monocytes to alternatively activated M2 macrophages is associated with the reorganization of the microtubule cytoskeleton. At lower concentrations, 2ME2 treatment depolymerized microtubules and reduced the expression of CD206 and CD163, suggesting that it inhibits the polarization of macrophages to M2 phenotype. However, the M1 polarization was not significantly affected at these concentrations. Importantly, 2ME2 inhibited the expression of several anti-inflammatory cytokines and growth factors, including CCL18, TGF-β, IL-10, FNT, arginase, CXCL12, MMP9, and VEGF-A, and hindered the metastasis-promoting effects of M2 macrophages. Concurrently, 2ME2 treatment reduced the expression of CD163 in tumors and inhibited lung metastasis in the orthotopic breast cancer model. Mechanistically, 2ME2 treatment reduced the phosphorylation and nuclear translocation of STAT3, an effect which was abrogated by colivelin.ConclusionsOur study presents novel findings on mechanism of 2ME2 from the perspective of its effects on the polarization of the TAMs via the STAT3 signaling in breast cancer. Altogether, the data supports further clinical investigation of 2ME2 and its derivatives as therapeutic agents to modulate the tumor microenvironment and immune response in breast carcinoma.

STING-Activating Polymer-Drug Conjugates for Cancer Immunotherapy.

The stimulator of interferon genes (STING) pathway links innate and adaptive antitumor immunity and therefore plays an important role in cancer immune surveillance. This has prompted widespread development of STING agonists for cancer immunotherapy, but pharmacological barriers continue to limit the clinical impact of STING agonists and motivate the development of drug delivery systems to improve their efficacy and/or safety. We developed SAPCon, a STING-activating polymer-drug conjugate platform based on strain-promoted azide-alkyne cycloaddition of a novel dimeric amidobenzimidazole (diABZI) STING prodrug to hydrophilic poly(dimethylacrylamide-co-azido-ethylmethacrylate) polymer chains through a cathepsin B-responsive linker to increase circulation time and enable passive tumor accumulation. We found that intravenously administered SAPCon accumulated at tumor sites, where it was endocytosed by tumor-associated myeloid cells, resulting in increased STING activation in the tumor tissue. Consequently, SAPCon promoted an immunogenic tumor microenvironment characterized by increased frequency of activated macrophages and dendritic cells and improved infiltration of CD8+ T cells, resulting in inhibition of tumor growth, prolonged survival, and enhanced response to anti-PD-1 immune checkpoint blockade in orthotopic breast cancer models. Collectively, these studies position SAPCon as a modular and programmable platform for improving the efficacy of systemically administered STING agonists for cancer immunotherapy.

Development and Characterization of Syngeneic Orthotopic Transplant Models of Obesity-Responsive Triple-Negative Breast Cancer in C57BL/6J Mice.

Obesity is an established risk and progression factor for triple-negative breast cancer (TNBC), but preclinical studies to delineate the mechanisms underlying the obesity-TNBC link as well as strategies to break that link are constrained by the lack of tumor models syngeneic to obesity-prone mouse strains. C3(1)/SV40 T-antigen (C3-TAg) transgenic mice on an FVB genetic background develop tumors with molecular and pathologic features that closely resemble human TNBC, but FVB mice are resistant to diet-induced obesity (DIO). Herein, we sought to develop transplantable C3-TAg cell lines syngeneic to C57BL/6 mice, an inbred mouse strain that is sensitive to DIO. We backcrossed FVB-Tg(C3-1-TAg)cJeg/JegJ to C57BL/6 mice for ten generations, and spontaneous tumors from those mice were excised and used to generate four clonal cell lines (B6TAg1.02, B6TAg2.03, B6TAg2.10, and B6TAg2.51). We characterized the growth of the four cell lines in both lean and DIO C57BL/6J female mice and performed transcriptomic profiling. Each cell line was readily tumorigenic and had transcriptional profiles that clustered as claudin-low, yet markedly differed from each other in their rate of tumor progression and transcriptomic signatures for key metabolic, immune, and oncogenic signaling pathways. DIO accelerated tumor growth of orthotopically transplanted B6TAg1.02, B6TAg2.03, and B6TAg2.51 cells. Thus, the B6TAg cell lines described herein offer promising and diverse new models to augment the study of DIO-associated TNBC.

Natural Derivatives of Selective HDAC8 Inhibitors with Potent in Vivo Antitumor Efficacy against Breast Cancer.

HDAC8 is a therapeutic target with great promise for breast cancer. Here, we reported a novel compound corallorazine D from Nocardiopsis sp. XZB108, selectively inhibited HDAC8 (IC50 = 0.90 ± 0.014 μM), suggesting that it may be a promising nonhydroxamate HDAC8 inhibitor. Upon additional modifications of corallorazine D, a candidate compound 5k, demonstrated remarkable inhibitory potency against HDAC8 (IC50 = 0.12 ± 0.01 nM), 89-fold superior to PCI-34051. The selectivity of 5k was at least 439-fold, superior to corallorazine D, confirming the efficacy of our modifications. In an orthotopic mouse model of breast cancer, 5k displayed nearly 4-fold superior antitumor activity than SAHA. Furthermore, 5k triggered antitumor immunity by activating T cells. Treatment with 5k significantly increased the proportion of M1 macrophages and decreased the proportion of M2 macrophages (M1/M2 ratio = 2.67 ± 0.25). 5k represents a promising compound for further investigation as a potential treatment for breast cancer.

Wedelolactone suppresses breast cancer growth and metastasis via regulating TGF-β1/Smad signaling pathway.

Breast cancer is a malignant tumor with high invasion and metastasis. TGF-β1-induced epithelial-mesenchymal transition (EMT) is crucially involved in the growth and metastasis of breast cancer. Wedelolactone (Wed) is extracted from herbal medicine Ecliptae Herba, which is reported to have antineoplastic activity. Here, we aimed to elucidate the efficacy and mechanism of Wed against breast cancer. The effects of Wed on migration and invasion of 4T1 were detected. The expression of EMT-related markers was detected by Western blot and qPCR. The 4T1 orthotopic murine breast cancer model was established to evaluate the therapeutic effect of Wed on the growth and metastasis of breast cancer through TGF-β1/Smad pathway. Wed inhibited the proliferation, migration and invasion of 4T1. It exhibited concentration-dependent inhibition of p-Smad2/3. Wed also reversed the expression of EMT-markers induced by TGF-β1. In addition, Wed suppressed the growth and metastasis of breast cancer in mice. It also affected p-Smad3 expression as well as EMT-related genes, suggesting that its anti-breast cancer effect may be related to the TGF-β1/Smad pathway. Wed reverses EMT by regulating TGF-β1/Smad pathway, potentially serving as a therapeutic agent for breast cancer. Wed is expected to be a potential drug to inhibit TGF-β1/Smad pathway-related diseases.

The F-actin bundler SWAP-70 promotes tumor metastasis.

Dynamic rearrangements of the F-actin cytoskeleton are a hallmark of tumor metastasis. Thus, proteins that govern F-actin rearrangements are of major interest for understanding metastasis and potential therapies. We hypothesized that the unique F-actin binding and bundling protein SWAP-70 contributes importantly to metastasis. Orthotopic, ectopic, and short-term tail vein injection mouse breast and lung cancer models revealed a strong positive dependence of lung and bone metastasis on SWAP-70. Breast cancer cell growth, migration, adhesion, and invasion assays revealed SWAP-70's key role in these metastasis-related cell features and the requirement for SWAP-70 to bind F-actin. Biophysical experiments showed that tumor cell stiffness and deformability are negatively modulated by SWAP-70. Together, we present a hitherto undescribed, unique F-actin modulator as an important contributor to tumor metastasis.

Synergistic effects of thermosensitive liposomal doxorubicin, mild hyperthermia, and radiotherapy in breast cancer management: an orthotopic mouse model study.

Liposome formulations of the cancer drug doxorubicin have been developed to address the severe side effects that result from administration of this drug in a conventional formulation. Among them, thermosensitive liposomal doxorubicin presents enhanced tumor targeting and efficient drug release when combined with mild hyperthermia localized to the tumor site. Exploiting the radiosensitizing benefits of localized thermal therapy, the integration of radiation therapy with the thermally activated liposomal system is posited to amplify the anti-tumor efficacy. This study explored a synergistic therapeutic strategy that combines thermosensitive liposomal doxorubicin, mild hyperthermia, and radiotherapy, using an orthotopic murine model of breast cancer. The protocol of sequential multi-modal treatment, incorporating low-dose chemotherapy and radiotherapy, substantially postponed the progression of primary tumor growth in comparison to the application of monotherapy at elevated dosages. Improvements in unheated distant lesions were also observed. Furthermore, the toxicity associated with the combination treatment was comparable to that of either thermosensitive liposome treatment or radiation alone at low doses. These outcomes underscore the potential of multi-modal therapeutic strategies to refine treatment efficacy while concurrently diminishing adverse effects in the management of breast cancer, providing valuable insight for the future refinement of thermosensitive liposomal doxorubicin applications.

The role of Nrf2 in redox coupling of tumor and adipose tissue in orthotopic breast cancer model

The role of Nrf2 in redox coupling of tumor and adipose tissue in orthotopic breast cancer model

Chronic High-Salt Diet Activates Tumor-Initiating Stem Cells Leading to Breast Cancer Proliferation.

Several chronic inflammatory diseases have been linked to high-salt (HS) diets. Chronic inflammation is an established causative hallmark of cancer. However, a direct role of HS diets in tumorigenesis is yet to be defined. Previous orthotopic murine breast tumor studies have shown that short-term HS diets caused inhibition of tumor growth through the activation of cytotoxic adaptive immune responses. However, there have been experimental challenges in developing a viable chronic HS-diet-based murine tumor model. To address this, we have developed a novel chronic HS diet tumor model through the sequential passaging of tumor cells in mice under HS dietary conditions. Two orthotopic murine triple-negative breast cancer models, 4T1 tumor cells injected into BALB/c mice and Py230 tumor cells injected into C57Bl/6 mice, were utilized in our study. For the HS diet cohort, prior to orthotopic injection with tumor cells, the mice were kept on a 4% NaCl diet for 2 weeks. For the regular salt (RS) diet cohort, the mice were kept on a 1% NaCl diet. Following syngeneic cancer cell injection, tumors were allowed to grow for 28 days, following which they were collected to isolate immune cell-depleted cancer cells (passage 1, P1). The tumor cells from P1 were reinjected into the next set of non-tumor-bearing mice. This procedure was repeated for three cycles (P2-P4). In P1, compared to the RS diet cohort, we observed reduced tumor kinetics in both murine tumor models on the HS diet. In contrast, by P4, there was significantly higher tumor progression in the HS diet cohort over the RS diet cohort. Flow cytometry analysis demonstrated an 8-fold increase in tumor-initiating stem cells (TISCs) from P1 to P4 of the HS diet cohort, while there were no significant change in TISC frequency with sequential passaging in the RS diet cohort. Molecular studies showed enhanced expression of TGFβR2 and CD80 on TISCs isolated from the P4 HS diet cohort. In vitro studies demonstrated that TGFβ stimulation of these TISCs increased the cellular expression of CD80 molecules. Further, the chronic HS diet selectively induced the glycolytic metabolic phenotype over the mitochondrial oxidative phosphorylation phenotype in TISCs, which is needed for the production of metabolites during tumor cell differentiation and proliferation. The infiltrating CD8 and CD4 T-lymphocytes in P4 tumors demonstrated increased expression of the immune checkpoint inhibitor (ICI) CTLA4, a known binding partner of CD80, to cause immune exhaustion and pro-tumorigenic effects. Interestingly, anti-TGFβ monoclonal antibodies (mAbs) played a synergistic role in further enhancing the anti-tumor effect of anti-CTLA4 mAb. In summary, our findings demonstrated that chronic HS diet increased the frequency of TISCs in tumors leading to blunting of cytotoxic adaptive immune responses causing tumor proliferation. Furthermore, a combination of anti-TGFβ with current ICI-based immunotherapies could exert more favorable anti-cancer clinical outcomes.

Self-Assembled Nanoparticles from the Amphiphilic Prodrug of Resiquimod for Improved Cancer Immunotherapy.

Tumor-associated macrophages (TAMs) usually adopt a tumor-promoting M2-like phenotype, which largely impedes the immune response and therapeutic efficacy of solid tumors. Repolarizing TAMs from M2 to the antitumor M1 phenotype is crucial for reshaping the tumor immunosuppressive microenvironment (TIME). Herein, we developed self-assembled nanoparticles from the polymeric prodrug of resiquimod (R848) to reprogram the TIME for robust cancer immunotherapy. The polymeric prodrug was constructed by conjugating the R848 derivative to terminal amino groups of the linear dendritic polymer composed of linear poly(ethylene glycol) and lysine dendrimer. The amphiphilic prodrug self-assembled into nanoparticles (PLRS) of around 35 nm with a spherical morphology. PLRS nanoparticles could be internalized by antigen-presenting cells (APCs) in vitro and thus efficiently repolarized macrophages from M2 to M1 and facilitated the maturation of APCs. In addition, PLRS significantly inhibited tumor growth in the 4T1 orthotopic breast cancer model with much lower systemic side effects. Mechanistic studies suggested that PLRS significantly stimulated the TIME by repolarizing TAMs into the M1 phenotype and increased the infiltration of cytotoxic T cells into the tumor. This study provides an effective polymeric prodrug-based strategy to improve the therapeutic efficacy of R848 in cancer immunotherapy.

Spatial transcriptomics reveals the interplay between cancer and immune cells directed by MXene quantum dots

Nanomedicine plays an essential role in the development of tumor treatment modalities. However, tumors have a complex structure that includes a variety of immune cell types, which, along with tumor cells, comprise the heterogeneous tumor microenvironment (TME). Although nanoparticles that overcome the limitations of other classical therapeutics and navigate heterogeneous biological barriers have been developed, many unknown players within the TME still exist. The role of immune cell populations and signaling pathways in the TME, which can affect the nanoparticle distribution in the tumor or the overall outcome of nanotherapeutic treatments, are poorly described. In this study, we used spatial transcriptomics to determine in situ gene expression and identified immune cells, other than tumor-associated macrophages, that play vital roles following nanoparticle exposure. In this proof-of-concept study, a recently explored type of nanoparticle, Ti3C2Tx MXene quantum dots (MQDs), with a single particle diameter of ≤10 nm, were administered to orthotopic breast cancer model mice. Thanks to their red-emitting fluorescence properties, we could track the distribution of MQDs in the tumor. Whole-transcriptome analysis and immunofluorescence staining suggested that the heterogeneous distribution of MQDs results in different tumor and immune cell responses in situ. We observed a more tumor-suppressive phenotype at tumor regions with high MQD accumulation compared to regions with decreased MQD accumulation or naïve tumors. Based on pathway analysis and cell deconvolution, we also identified other immune cell types altered by MQDs, including B cells and neutrophils. Specifically, the application of MQDs recruited and activated B cells and resulted in neutrophil degranulation and NETosis in vivo. Using spatial transcriptomics technology, we defined the fundamental molecular and cellular changes that occur in situ in the TME following MQD administration. Future spatial omics studies involving different nanoparticles with other material characteristics will help design more effective nanotherapeutics.

Residual OXPHOS is required to drive primary and metastatic lung tumours in an orthotopic breast cancer model.

Fast adaptation of glycolytic and mitochondrial energy pathways to changes in the tumour microenvironment is a hallmark of cancer. Purely glycolytic ρ0 tumour cells do not form primary tumours unless they acquire healthy mitochondria from their micro-environment. Here we explored the effects of severely compromised respiration on the metastatic capability of 4T1 mouse breast cancer cells. 4T1 cell lines with different levels of respiratory capacity were generated; the Seahorse extracellular flux analyser was used to evaluate oxygen consumption rates, fluorescent confocal microscopy to assess the number of SYBR gold-stained mitochondrial DNA nucleoids, and the presence of the ATP5B protein in the cytoplasm and fluorescent in situ nuclear hybridization was used to establish ploidy. MinION nanopore RNA sequence analysis was used to compare mitochondrial DNA transcription between cell lines. Orthotopic injection was used to determine the ability of cells to metastasize to the lungs of female Balb/c mice. OXPHOS-deficient ATP5B-KO3.1 cells did not generate primary tumours. Severely OXPHOS compromised ρ0D5 cells generated both primary tumours and lung metastases. Cells generated from lung metastasis of both OXPHOS-competent and OXPHOS-compromised cells formed primary tumours but no metastases when re-injected into mice. OXPHOS-compromised cells significantly increased their mtDNA content, but this did not result in increased OXPHOS capacity, which was not due to decreased mtDNA transcription. Gene set enrichment analysis suggests that certain cells derived from lung metastases downregulate their epithelial-to-mesenchymal related pathways. In summary, OXPHOS is required for tumorigenesis in this orthotopic mouse breast cancer model but even very low levels of OXPHOS are sufficient to generate both primary tumours and lung metastases.

Tumor Oxygenation in an Orthotopic Model of Breast Cancer: Impact of Dietary Nitrate Supplementation

Introduction: Breast cancer (BC), the predominant non-melanoma cancer among women, is projected to escalate to more than 3 million new cases and ~1 million fatalities globally by 2040. Chronic and fluctuating low oxygen (O2) conditions within tumors favor malignant growth, decreased responsiveness to therapeutic interventions, and reduced patient survivability. Treatment strategies to combat tumor hypoxia are desperately needed. Dietary nitrate supplementation via beetroot juice (BRJ), which increases nitric oxide (NO) production through the nitrate-nitrite-NO pathway, has been demonstrated to increase skeletal muscle blood flow, improve mitochondrial effciency, and reduce metabolic demands. Specifically, BRJ improves interstitial oxygen pressures (PO2 is) in acidic, low oxygen environments such as that found in the tumor microenvironment. Therefore, we hypothesized that BRJ supplementation would increase PO2 is in BC tumors. Methods: Female Fischer 344 retired breeder rats (6-8 months old) were used in this investigation. Adenocarcinoma cells (MAT B III at 6 x 103) were injected into the mammary duct and tumor growth was monitored over 2 weeks. Once tumors reached ~5 mm in diameter, rats were randomly assigned to consume BRJ (1 mmol/kg/day; BRJ, n=3) or water (CON; n=3) for 5 days. Under pentobarbital anesthesia, tumor PO2 is measurements were obtained using phosphorescence quenching. Results: Tumor growth rate was decreased in BRJ compared to CON rats (0.5 ± 0.1 vs. 1.0 ± 0.1 mm/day; p<0.05). Heart rate and mean arterial pressure were not different between groups (P>0.05). Blood lactate was significantly decreased in BRJ compared to CON rats (0.5 ± 0.1 vs. 1.2 ± 0.1 mmol/L, p<0.05). There was an increase in mean tumor PO2 is in BRJ vs. control rats (6.9 ± 1.3 vs. 3.7 ± 0.47; p<0.05). Conclusions: This study offers promising preliminary insights into the potential of dietary nitrate supplementation as an adjunctive approach to BC treatment to increase tumor PO2 is and reduce tumor growth rate. Future and ongoing studies will increase the sample size, use fluorescent microspheres to measure tumor blood flow, and explore the potential for BRJ and other NO agonists to combat tumor hypoxia and the skeletal muscle dysfunction associated with cancer progression. This research was funded by the following sources: the Johnson Cancer Research Center Innovative Research Award and Sustained Momentum for Investigators with Laboratories Established (SMILE) Grants at Kansas State University, the National Institute on Aging (Grant 1R15AG078060), and Ruth L. Kirschstein National Research Service Awards from the National Heart, Lung, and Blood Institute (Grants 1F31HL167618-01 and 1F31HL170643-01). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Nanoparticle Retinoic Acid-Inducible Gene I Agonist for Cancer Immunotherapy.

Pharmacological activation of the retinoic acid-inducible gene I (RIG-I) pathway holds promise for increasing tumor immunogenicity and improving the response to immune checkpoint inhibitors (ICIs). However, the potency and clinical efficacy of 5'-triphosphate RNA (3pRNA) agonists of RIG-I are hindered by multiple pharmacological barriers, including poor pharmacokinetics, nuclease degradation, and inefficient delivery to the cytosol where RIG-I is localized. Here, we address these challenges through the design and evaluation of ionizable lipid nanoparticles (LNPs) for the delivery of 3p-modified stem-loop RNAs (SLRs). Packaging of SLRs into LNPs (SLR-LNPs) yielded surface charge-neutral nanoparticles with a size of ∼100 nm that activated RIG-I signaling in vitro and in vivo. SLR-LNPs were safely administered to mice via both intratumoral and intravenous routes, resulting in RIG-I activation in the tumor microenvironment (TME) and the inhibition of tumor growth in mouse models of poorly immunogenic melanoma and breast cancer. Significantly, we found that systemic administration of SLR-LNPs reprogrammed the breast TME to enhance the infiltration of CD8+ and CD4+ T cells with antitumor function, resulting in enhanced response to αPD-1 ICI in an orthotopic EO771 model of triple-negative breast cancer. Therapeutic efficacy was further demonstrated in a metastatic B16.F10 melanoma model, with systemically administered SLR-LNPs significantly reducing lung metastatic burden compared to combined αPD-1 + αCTLA-4 ICI. Collectively, these studies have established SLR-LNPs as a translationally promising immunotherapeutic nanomedicine for potent and selective activation of RIG-I with the potential to enhance response to ICIs and other immunotherapeutic modalities.

Abstract LB123: Conditionally active, therapeutic lymphotoxin beta receptor (LTBR) agonist bispecific antibodies for induction of tertiary lymphoid structures in the treatment of solid tumors

Abstract The presence of tertiary lymphoid structures (TLS) and associated high endothelial venules (HEV) in the tumor microenvironment strongly correlates with improved prognosis and treatment outcomes across a wide range of solid tumors. These clinical observations, now replicated repeatedly, highlight the therapeutic potential of induction of TLS and/or boosting TLS functions, which include acting as a point of entry and education for immune effector cells locally in the tumor. Lymphotoxin beta receptor (LTBR) signalling is essential for lymphoid structure development and the ectopic expression of its ligands, lymphotoxin αβ and LIGHT, is sufficient for TLS formation in vivo but challenging for therapeutic development. Here we present human bispecific antibodies (bsAbs) which conditionally agonize LTBR on co-engagement of fibroblast activation protein (FAP), a tumor microenvironment specific marker expressed by cancer associated fibroblasts. In vitro characterisation of these therapeutic bsAbs is presented, along with in vivo preclinical evaluation of surrogate bsAbs binding to mouse LTBR and mouse FAP in murine tumor models. Results - Therapeutic bsAbs were identified following an extensive screening campaign combining LTBR and FAP binding arms on an engineered human Fc with minimal FcyR binding. In vitro assays using primary cancer associated fibroblasts and LTBR positive cells demonstrated conditionally active therapeutic bsAbs potently activated LTBR in the presence of FAP expressing cells, whereas in the absence of FAP expressing cells no activity was observed. Surrogate bsAbs demonstrated monotherapy activity and led to tumor regressions in combination with anti-PDL1 therapy in an EMT6 mouse model, a syngeneic orthotopic model of breast cancer. Flow cytometry of endpoint tumors showed robust formation of HEVs and increased infiltration of T cells and B cells. In addition, we demonstrate that LTBR agonism, but not PD-L1 inhibition, leads to the formation of TLS structures containing organized lymphocyte aggregates with the appearance of germinal centres and accumulations of T and B cells in a mouse model of lung cancer. In conclusion, conditionally active FAP-LTBR bispecifics activate LTBR in the tumor microenvironment and induce HEV and TLS formation, leading to potent monotherapy activity in vivo, and to tumor regression in combination with PD-L1. These data support the development of FAP-LTBR bispecifics for the treatment of solid tumors as monotherapy and in combination with standard of care. Citation Format: Marta Lewandowska, Fevzi Demircioglu, Lucy Penfold, Rebecca B. Riddle, Sameer Sirohi, Floriane Laurent, Richard Brown, Sonia Bains, Dara Bevan, Emma Stanley, Jeanine Pignatelli, James W. Legg, Ray Jupp. Conditionally active, therapeutic lymphotoxin beta receptor (LTBR) agonist bispecific antibodies for induction of tertiary lymphoid structures in the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB123.

Abstract LB034: First-in-class compounds targeting the MIEN1 signaling pathway: A breakthrough approach for breast and prostate cancer therapy

Abstract We report the identification and complete characterization of first-in-class molecules targeting the MIEN1 signaling pathway in cancer. Migration and invasion enhancer 1 (MIEN1) overexpression characterizes several cancers and facilitates cancer cell migration and invasion. Leveraging conserved ITAM and prenylation motifs within MIEN1, we identified potent anti-cancer peptides. Among them, bioactive peptides LA3IK and RP-7 induced pronounced transcriptomic and protein expression changes at sub-IC50 concentrations. The peptides effectively inhibited genes and proteins driving cancer cell migration, invasion, and EMT pathways, concurrently suppressing EGF-induced NF-kB nuclear translocation in metastatic breast cancer cells. Specifically, peptides targeted the same signal transduction pathway initiated by MIEN1. Molecular docking and circular dichroism spectroscopy indicated the formation of MIEN1-peptide complexes. The third-positioned isoleucine in LA3IK and CVIL motif in RP-7 were crucial for inhibiting breast cancer cell migration. This is evident from the limited migration inhibition observed when MDA-MB-231 cells were treated with scrambled peptides LA3IK-SCR and RP-7-SCR. Additionally, LA3IK and RP-7 effectively suppressed tumor growth in an orthotopic breast cancer model. Notably, mice tolerated high peptide doses of up to 90 mg/Kg well, surpassing significantly lower doses of 5 mg/Kg intravenously (iv) and 30 mg/Kg intraperitoneally (ip) used in both in vivo pharmacokinetic studies and orthotopic mouse model assays. D-isomers of LA3IK and RP-7 showed enhanced anti-cancer activity compared to their L-isomers. D-LA3IK remained stable in mouse plasma for 24 h with 75% remaining, exhibiting superior pharmacokinetic properties over D/L-RP-7. In summary, our findings mark the first report of short peptides based on MIEN1 protein sequence capable of inhibiting cancer signaling pathways, effectively impeding cancer progression both in vitro and in vivo. Citation Format: Amit Kumar Tripathi, Jamboor K. Vishwanatha. First-in-class compounds targeting the MIEN1 signaling pathway: A breakthrough approach for breast and prostate cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB034.

Abstract LB355: Cryo-immune vaccination (CIV) by SYNC-T therapy: Preclinical modeling of a novel device-multidrug immunotherapeutic approach to eradicate advanced metastatic cancers

Abstract A major challenge to further development of immune checkpoint therapies (ICT) for advanced cancer is the inherent toxicity of higher-order multidrug combinations likely needed to eradicate metastatic disease. Recently, early clinical studies have investigated a new general approach to address this challenge, termed cryo-immune vaccination (CIV). The CIV approach by SYNC-T therapy involves cycles of local cryolysis of a solid tumor followed by infusion of the same lesion with a low-dose multi-API formulation. CIV has never been explored methodically in preclinical or clinical settings, so the basis for systemic responses that have been observed in early clinical testing are not fully understood. Accordingly, as trials of the safety and efficacy of CIV proceed, there is now a need of preclinical model systems to help illuminate the mechanistic foundations of the approach, investigate safety and efficacy, and learn where and how it might be best leveraged. To develop the conceptual framework of CIV, we adapted the orthotopic metastatic 4T1 breast cancer model used extensively in our laboratory as a foundation to study CIV antitumor responses and mechanisms of action. Briefly, breast tumors were initiated in naïve murine subjects by orthotopic injection of 1x104 cells into the mammary fad pads of immunocompetent BALB/c hosts. When tumors reached ~5-7 mm, a time when occult pulmonary metastases have seeded, primary tumors were exposed surgically on skin flaps and contacted directly with a liquid nitrogen-cooled pointed steel rod for 15-30 sec. The rod was then withdrawn and tumor tissue allowed to thaw fully before injection into the cryolysed tumor of a selected ICT multidrug cocktail (or negative control murine IgG) in 25-50 µl total volume. The selected multidrug cocktail was modeled on SYNC-T combination therapy currently under study in a Phase 1 clinical trial (Syncromune, Inc.) Briefly, the ICT drug cocktails were variably composed of murine-reactive antibodies against CTLA4, PD-1 or OX40 (agonist), CD40L (agonist) and CpG oligonucleotide. As a starting point, allometric scaling was used to model dosing analogous to the proprietary ICT drug formulations SV-101 and SV-102 in clinical testing (Syncromune, Inc.). For exploratory toxicology, a standard panel of blood-borne liver and kidney toxicity biomarkers was assessed. For exploratory pharmacology, a ‘super mouse’ design was employed with ELISA-based determinations to measure antibody distribution in blood serum. To gain initial mechanistic insights, responses will be compared in host mice that are genetically deficient in selected components of the adaptive or innate immune systems. Progress in methodological and mechanistic studies will be presented. Citation Format: James B. DuHadaway, Alexander J. Muller, Lisa D. Laury-Kleintop, U. Margaretha Wallon, Susan K. Gilmour, Marie Webster, Jason R. Williams, Gabriela R. Rossi, Mario R. Mautino, Jonathan Lewis, Charles J. Link, George C. Prendergast. Cryo-immune vaccination (CIV) by SYNC-T therapy: Preclinical modeling of a novel device-multidrug immunotherapeutic approach to eradicate advanced metastatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB355.