Bearing Mice Research Articles

Development of a novel immunocompetent murine tumor model for urothelial carcinoma using in vivo electroporation

A lack of advanced preclinical mouse tumor models impedes the progress in urothelial carcinoma research. We present here a novel fast, robust, reliable, and highly reproducible model for the genetic induction of bladder cancer in immunocompetent mice. Different sets of oncogenic transposons (Cmyc, Kras) and Cre drivers were transfected into the murine bladder wall of two different genetic backgrounds (Trp53fl/fl and BrafV600E, Ptenfl/fl, Ctnnb1exon3-fl/fl). Transfection was carried out using in vivo electroporation of the bladder after surgical exploration and transmural or transurethral intravesical plasmid injection. Up to 100% of animals developed urothelial carcinomas of the bladder. Time to tumor onset ranged from 16 to 97 days with a median of approximately 23 days in the fastest groups. Histological examination identified orthotopic urothelial carcinomas in most cases, in some experimental groups up to 100%. The resulting tumors were highly invasive and often metastatic. Metastases were found in up to 100% of tumor bearing mice per group. Taken together, this study establishes the proof-of-principle that in vivo electroporation can be versatilely employed as a reliable, fast, and robust method for the highly reproducible induction of urothelial carcinomas in the murine bladder wall. This novel murine tumor model could pave the way towards more easily modelling subtype specific urothelial carcinomas in mice.

Combining ERAP1 silencing and entinostat therapy to overcome resistance to cancer immunotherapy in neuroblastoma

BackgroundCheckpoint immunotherapy unleashes tumor control by T cells, but it is undermined in non-immunogenic tumors, e.g. with low MHC class I expression and low neoantigen burden, such as neuroblastoma (NB). Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an enzyme that trims peptides before loading on MHC class I molecules. Inhibition of ERAP1 results in the generation of new antigens able of inducing potent anti-tumor immune responses. Here, we identify a novel non-toxic combinatorial strategy based on genetic inhibition of ERAP1 and administration of the HDAC inhibitor (HDACi) entinostat that increase the immunogenicity of NB, making it responsive to PD-1 therapy.MethodsCRISPR/Cas9-mediated gene editing was used to knockout (KO) the ERAP1 gene in 9464D NB cells derived from spontaneous tumors of TH-MYCN transgenic mice. The expression of MHC class I and PD-L1 was evaluated by flow cytometry (FC). The immunopeptidome of these cells was studied by mass spectrometry. Cocultures of splenocytes derived from 9464D bearing mice and tumor cells allowed the assessment of the effect of ERAP1 inhibition on the secretion of inflammatory cytokines and activation and migration of immune cells towards ERAP1 KO cells by FC. Tumor cell killing was evaluated by Caspase 3/7 assay and flow cytometry analysis. The effect of ERAP1 inhibition on the immune content of tumors was analyzed by FC, immunohistochemistry and multiple immunofluorescence.ResultsWe found that inhibition of ERAP1 makes 9464D cells more susceptible to immune cell-mediated killing by increasing both the recall and activation of CD4+ and CD8+ T cells and NK cells. Treatment with entinostat induces the expression of MHC class I and PD-L1 molecules in 9464D both in vitro and in vivo. This results in pronounced changes in the immunopeptidome induced by ERAP1 inhibition, but also restrains the growth of ERAP1 KO tumors in vivo by remodelling the tumor-infiltrating T-cell compartment. Interestingly, the absence of ERAP1 in combination with entinostat and PD-1 blockade overcomes resistance to PD-1 immunotherapy and increases host survival.ConclusionsThese findings demonstrate that ERAP1 inhibition combined with HDACi entinostat treatment and PD-1 blockade remodels the immune landscape of a non-immunogenic tumor such as NB, making it responsive to checkpoint immunotherapy.

Abstract A031: Unhelpful CD4 help: Removal of conventional CD4+ T cells promotes priming of tumor antigen-specific CD8 T cells in the context of CD4-depletion therapy

Abstract Preclinical and clinical studies have used antibodies to deplete CD4-expressing cells to promote anti-tumor immunity. Specifically, a monoclonal anti-CD4 antibody (anti-CD4) has been employed to remove FOXP3+ CD4+ regulatory T cells (Tregs), which are known to impede anti-tumor immunity. Our previous studies demonstrated that administering anti-CD4 during B16 melanoma tumor growth in mice leads to the generation of tumor Ag-specific CD8 T cells that disseminate and form memory following surgical tumor excision. However, the importance of co-depleting other CD4-expressing cell compartments for anti-CD4 treatment efficacy are unknown. We utilized flow cytometry and single-cell RNA sequencing (scRNAseq) to evaluate the primary response of tumor Ag-specific CD8 T cells (pmel) in B16 melanoma tumor bearing mice treated with anti-CD4. Responses were compared to those induced by anti-PD1 and anti-CTLA4 dual immune checkpoint blockade (ICB). To determine the importance of homeostatic space in priming, homeostatic expansion and priming were decoupled, and neutralizing antibodies were administered. Treg-depleted (Foxp3-DTR) mice were used for comparison, and Batf3-/- mice and IL-2R blocking antibodies were used to determine requirements for CD8 T cell priming. Finally, scRNAseq was conducted on CD4 T cells from human melanoma tumors previously treated with ICB to characterize transcriptional properties of intratumoral CD4 T cells. Compared to ICB, anti-CD4 treatment elicited a more robust, disseminated, and persistent primary tumor Ag-specific CD8 T cell response. ScRNAseq revealed that anti-CD4 treatment induced more stem-like (TCF1+) memory precursor cells, whereas ICB induced more differentiated effector (Gzmb+) cells. Removal of homeostatic space or blockade of IL-7R and IL-15 during priming did not diminish pmel responses in anti-CD4 treated mice, indicating that homeostatic space was not required. Importantly, pmel cells were shown to produce their own IL-2, and pmel priming was significantly lower after IL-2 receptor blockade, suggesting dependence on autocrine IL-2 signals. Using DT-treated FOXP3-DTR mice, we found that targeted Treg depletion was insufficient for the pmel priming that disseminated and formed memory, indicating that total CD4 T cell depletion was required for this systemic response. Depletion of total CD4 T cells uniquely increased cDC1 proportions in tumor-draining LNs as well as their expression of CD86. However, treatment of tumor-bearing RAG-/- mice with anti-CD4 confirmed that depletion of CD4-expressing APC compartments does not contribute to anti-CD4 treatment efficacy. ScRNAseq of CD4 T cells from human melanoma tumors showed dominance of Tr1-like cells and other subsets without transcriptional evidence of helper differentiation, despite prior ICB. In mice, combination of anti-CD4 with dual ICB restored the systemic response of tumor-specific CD8 T cells. This study underscores the importance of eliminating conventional CD4 T cells in tumor-bearing hosts to elicit systemic primary and memory CD8 T cell responses. Citation Format: Christo P. C. Dragnev, Delaney E. Ramirez, Tyler G. Searles, Nathaniel Spicer, TIffany Chen, J. Louise Lines, Aaron R. Hawkes, Wilson L. Davis III, Asmaa Mohamed, Keisuke Shirai, Joesph D. Phillips, Pamela C. Rosato, Yina H. Huang, Mary Jo Turk. Unhelpful CD4 help: Removal of conventional CD4+ T cells promotes priming of tumor antigen-specific CD8 T cells in the context of CD4-depletion therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A031.

Targeted nanoliposomes of oncogenic protein degraders: Significant inhibition of tumor in lung-cancer bearing mice

With 60 % of non-small cell lung cancer (NSCLC) expressing epidermal growth factor receptor (EGFR), it has been explored as an important therapeutic target for lung tumors. However, even the well-established EGFR inhibitors tend to promptly develop resistance over time. Moreover, strategies that could impede resistance development and be advantageous for both EGFR-Tyrosine kinase inhibitor (TKI)-sensitive and mutant NSCLC patients are constrained. Based on the critical relationship between EGFR, c-MYC, and Kirsten rat sarcoma virus (K-Ras), simultaneous degradation of EGFR and Bromodomain-containing protein 4 (BRD4) using “Proteolysis Targeting Chimeras (PROTACs)” could be a promising approach. PROTACs are emerging class of oncoprotein degraders but very challanging to deliver in vivo. Compared to individual IC50s, strong synergism was observed at 1:1 ratio of BPRO and EPRO in NSCLC cell lines with diverse mutation. Significant inhibition of cell growth with higher cellular apoptosis was observed in 2D and 3D-based cell assays in nanomolar concentrations. EGFR activation assay revealed 47.60 % EGFR non-expressing cells confirming EGFR-degrading potential of EPRO. A lung cancer specific nanoliposomal formulation of EGFR and BRD4-degrading PROTACs (EPRO and BPRO) was prepared and characetrized. Successful encapsulation of the two highly lipophilic molecules was achieved in EGFR-targeting nanoliposomal carriers (T-BEPRO) using a modified hydration technique. T-BEPRO revealed a particle size of 109.22 ± 0.266 nm with enhanced cellular uptake and activity. Remarkably, parenterally delivered T-BEPRO in tumor-bearing mice showed a substantially higher % tumor growth inhibition (TGI) of 77.6 % with long-lasting tumor inhibitory potential as opposed to individual drugs.

ESOMA-DM1, a Maytansinoid-Based Theranostic Small-Molecule Drug Conjugate for Neuroendocrine Tumors.

Background: The main challenges of conventional chemotherapy lie in its lack of selectivity and specificity, leading to significant side effects. Using a small-molecule drug conjugate (SMDC) ensures specific delivery of a cytotoxic drug to the tumor site by coupling it to a targeting vector. This promising strategy can be applied to neuroendocrine tumors (NETs) by choosing a targeting vector that binds specifically to somatostatin receptor subtype 2 (SSTR2). Additionally, incorporation of a bifunctional chelate into the molecule enables complexation of both diagnostic and therapeutic radionuclides. Thus, it facilitates monitoring of the distribution of the SMDC in the body and allows for the implementation of combination therapy. In our study, we designed eSOMA-DM1, a SMDC combining the SSTR2-targeted octreotate peptide and the cytotoxic agent DM1 via a chelate-bridged linker (N3-Py-DOTAGA). This approach warrants conjugation of the targeting vector and the drug at opposite sites to avoid undesired steric hindrance effects. Methods: Synthesis of the DM1 moiety (4) involved a three-step synthetic route, followed by the conjugation to the cyclic peptide, N3-Py-DOTAGA-d-Phe-cyclo[Cys-Tyr-d-Trp-Lys-Thr-Cys]-Thr-OH, through a copper-free click reaction, resulting in eSOMA-DM1. Subsequent labeling with [111In]InCl3 gave a high radiochemical yield and purity. In vitro assessments of eSOMA-DM1 binding, uptake, and internalization were conducted in SSTR2-transfected U2OS cells. Ex vivo biodistribution and fluorescence imaging were performed in H69-tumor bearing mice. Results: eSOMA-DM1 exhibited an IC50 value for SSTR2 similar to the gold standard DOTA-TATE. The uptake of [111In]In-eSOMA-DM1 in U2OS.SSTR2 cells was 1.2-fold lower than that of [111In]In-DOTA-TATE. Tumor uptake in H69-xenografted mice was higher for [111In]In-eSOMA-DM1 at all-time points compared to [111In]In-DOTA-TATE. Prolonged blood circulation led to increased accumulation of [111In]In-eSOMA-DM1 in highly vascularized tissues, such as the lungs, skin, and heart. Excretion through the kidneys, liver, and spleen was also observed. Conclusion: eSOMA-DM1 is a SMDC developed for NET showing promising characteristics in vitro. However, the in vivo results obtained with [111In]In-eSOMA-DM1 suggest the need for adjustments to optimize its distribution.

Construction of in-situ self-assembled agent for NIR/PET dual-modal imaging and photodynamic therapy for hepatocellular cancer

Hepatocellular cancer (HCC) remained a life-threatening carcinoma. Agents for HCC imaging and therapy were expected to possess different intratumoral retention time. To construct an agent with different intratumoral retention time when applied for tumor imaging or therapy remained great values. A lasialoglycoprotein receptor (ASGPR) targeted lactobionic acid derivative (LABO) was constructed for fluorescent imaging and photodynamic therapy of HCC. 18F labeled LABO (18F-LABO) was developed for PET imaging of HCC. LABO and 18F-LABO showed similar molecular structure. LABO exhibited characteristic of viscosity and concentration-induced intratumoral in-situ self-assembly to expand the intratumoral retention. LABO was non-fluorescent at free stage, but emitted NIR fluorescence and generated irradiation-induced ROS after self-assembly for fluorescent imaging and photodynamic therapy. ASGPR specificity of LABO and 18F-LABO was confirmed using HepG2 cell. Biodistribution and fluorescent imaging confirmed the different tumor retention time of LABO and 18F-LABO when used for photodynamic therapy and PET imaging. PET imaging and photodynamic therapy were performed on HepG2 tumor bearing mice, which revealed that 18F-LABO/LABO could specifically accumulated in the HepG2 tumor for tumor location/inhibition. LABO/18F-LABO with excellent HCC specificity but different intratumoral behaviors showed great values for the PET/NIR imaging and photodynamic therapy for HCC.Graphical

Automated segmentation pipeline for segmentation and characterising metabolic dysregulation in cancer cachexia

Cancer cachexia disrupts metabolic homeostasis across various organs. Positron Emission Tomography (PET) is valuable for investigating cachexia in preclinical models and patients[1]. However, manually segmenting multiple organs is laborious and hinders quantitative analysis of whole-body PET. This study establishes an automated pipeline for segmenting and characterizing organs in preclinical PET/MRI. Acute metabolic changes were induced in non-tumour bearing mice through three approaches: a single dose of Growth Differentiation Factor-15 (GDF-15; n=3 treated, control), daily dexamethasone for 24 days (n=4 treated, control), and 20 hour fasting (n=2 fasted, n=2 fed). [18F]flurodeoxy-glucose PET was used to image whole-body glucose utilisation. To overcome manual segmentation limitations, an automated multi-organ segmentation method was developed for preclinical MRI using nnU-net, an artificial intelligence architecture[2,3]. The nnU-net was trained on 18 preclinical MRI scans with 21 manual tissue annotations. Evaluation on 8 additional mice revealed high accuracy (Dice Coefficients: 0.72-0.91 for 17 tissues). Adipose tissues had lower accuracy but retained good specificity. Due to low accuracy, pancreas segmentation was excluded. All mice used for training and testing the nnU-net were imaged with the same protocol, sourced from the described datasets, with additional scans from different time points. After segmenting the scans, we measured mean Standardized Uptake Values (quantitative measure of metabolic activity) for each tissue. Significant differences (p<0.05) were observed following the various interventions (GDF-15, dexamethasone, fasting) compared to control. Network analysis provided insights into potential inter-organ interactions, while Linear Discriminant Analysis successfully distinguished between each metabolic state. This study presents a novel method for automated segmentation and analysis within preclinical PET/MRI. This method significantly improves efficiency and provides new analysis of total-body metabolic phenotypes. We are currently applying this approach to explore metabolic changes associated with cancer cachexia, aiming to improve our understanding of systemic metabolic dysregulation in both preclinical models and clinical settings. Please click on the 'PDF' for the full abstract!

Sirtinol, a SIRT1 inhibitor, inhibits the EMT and metastasis of 4T1 breast cancer cells and impacts the tumor microenvironment.

The impact of epigenetic drugs on metastasis and the immunological microenvironment is poorly understood. In this study, we looked at how sirtinol, a SIRT1 inhibitor, affected epithelial-mesenchymal transition (EMT), metastasis, and the immune cells. In vitro experiments were carried out using tumor conditioned medium (TCM). For in vivo experiments, sirtinol was administered i.p. in tumor bearing BALB/c mice at a dose of 2 mg/kg body weight either alone or in combination with cisplatin. Estimation of cytokines was carried out using ELISA or ELIspot. Estimation of different markers was done using flow cytometry or western blot. Sirtinol, a SIRT1 inhibitor, was found to be cytotoxic to 4T1 breast cancer cells with no synergistic effects with cisplatin, both under in vitro and in vivo conditions (p < 0.05). Sirtinol significantly reduced cancer cell metastasis to the spleen which was supported by in vitro findings such as decreased vimentin expression and cell mobility in migration and wound healing assays (p < 0.01). Studies on the effects of 4T1 tumor-conditioned medium on spleen cells indicated changes in T cell proliferation as well as differentiation (p < 0.01). In tumor bearing mice, spleen cells showed elevated IFN-γ secretion, increased CD11b+ cells, and decreased T cells (p < 0.01). This was reversed by sirtinol as well as the combination treatment, which may also have contributed to metastasis inhibition (p < 0.01). Sirtinol, a SIRT1 inhibitor inhibits EMT and metastasis of 4T1 breast cancer cells and also has an impact on the immune microenvironment.

Mechanism of Shashen-Maidong herb pair in treating hepatocellular carcinoma using network pharmacology and experimental validation

Ethnopharmacological relevanceHepatocellular carcinoma (HCC) is among the most prevalent malignant tumors globally and represents a significant public health issue worldwide. Immune cell dysfunction is the crucial factor for the formation of immunosuppression microenvironment of HCC. Glehnia littoralis (A.Gray) F.Schmidt ex Miq. (Shashen) and Ophiopogon japonicus (Thunb.) Ker Gawl. (Maidong) are classic herb pair in traditional Chinese medicine (TCM) of nourishing Yin, and is widely applied in the treatment of HCC and possesses multiple immunomodulatory functions. However, the role of the the Shashen-Maidong herb pair (SS-MD) for the management of HCC and the potential mechanisms has not been explicated. Aim of the studyThe purpose of the research is to investigate the potential mechanism of the SS-MD herb pair for the management of HCC. Materials and methodsThe known components of the SS-MD herb pair were preliminarily identified using UPLC-Q-Orbitrap-MS/MS. The active ingredients of SS-MD herb pair in treating HCC were screened by constructing herb-component-target network, and the key therapeutic targets were explored by constructing a protein-protein interaction (PPI) network. The binding affinity of the key targets and components were validated through molecular docking and molecular dynamics simulations. GO biological function and KEGG pathway analyses were operated to elucidate the potential mechanisms of the SS-MD herb pair for the management of HCC. And the mechanism was verified in the tumor bearing mice model and cell co-culture experiments. ResultsNetwork pharmacology prediction revealed 39 active components and 138 targets of the SS-MD herb pair for the treatment of HCC. KEGG analysis mainly focused on Notch signaling pathway and Apoptosis signaling pathway. The targets were enriched in biological functions of lymphocyte effector function and lymphocyte apoptosis. In vivo and in vitro experiments proved that the SS-MD herb pair can improve the proportion of CD8+T cells in the HCC immune microenvironment, regulate its subgroup distribution. SS-MD herb pair promoted CD8+T cells to secrete IL-2, TNF-α, IFN-γ, Granzyme B and Perforin, and inhibited apoptosis by regulating Notch signaling pathway. ConclusionsThis study identified the key components, targets, and signaling pathways of the SS-MD herb pair, confirm that SS-MD herb pair play an immunomodulatory role in treating HCC, provides theoretical support for the collaborative treatment of HCC with TCM.

Construction of DNA Vaccine of Extracellular Region of Vascular Endothelial Growth Factor Receptor 2 and Its Antitumor Activity in Vivo

Introduction: it constructed recombinant DNA vaccine of extracellular region of vascular endothelial growth factor receptor 2 (VEGFR2) and analyzed its in vivo activity against H22 transplanted hepatoma. Methods: the gene fragment of VEGFR2 extracellular region 1-3 was amplified, and the pcDNA3.1(+)/exVEGFR2 recombinant plasmid was constructed. The expression of exVEGFR2 was detected by Western blot. Subsequently, liposome (LP) pcDNA3.1(+)/exVEGFR2 complex (LP-pcDNA3.1(+)/exVEGFR2) was prepared and immunized to C57BL/6 mice. The cytotoxic activity mediated by cytotoxic T cells (CTLs) was analyzed by 51Cr release assay. H22 tumor bearing C57BL/6 mouse model was prepared. Quadriceps femoris muscle was injected with normal saline (saline group, AG), LP-pcDNA3.1(+) 100 μL (1 μg/μL, LP-pcDNA3.1(+) group, BG), and LP-pcDNA3.1(+)/exVEGFR2 100 μL (1 μg/μL, LP-pcDNA3.1(+)/exVEGFR2 group, CG). Tumor growth and death were recorded, and microvessel density (MVD) was evaluated by CD31 immunohistochemical staining. Results: a 1252 bp of exVEGFR2 sequence was amplified, and a specific band expressing 44 kDa molecular weight was constructed by transfecting pcDNA3.1(+)/exVEGFR2 into COS-7 cells. After immunizing C57BL/6 mice for 6 weeks, CTLs experiment suggested that LP-pcDNA3.1(+)/exVEGFR2 could effectively mediate the toxic effect of VEGFR2 positive H22 cells. The in vivo antitumor activity experiment found that as against AG and BG, the tumor volume, weight, and MVD were markedly reduced, the tumor latency time was markedly prolonged, and the average survival time was also markedly prolonged in CG (P &lt; 0.05). Conclusion: LP-pcDNA3.1(+)/exVEGFR2 can effectively activate C57BL/6 mice to produce specific anti-VEGFR2 immune response, and then produce anti-tumor cell immunotoxicity in vitro and in vivo. Inhibiting angiogenesis can effectively prolong the survival time of H22 tumor bearing mice.

Brusatol improves the efficacy of sorafenib in Huh7 cells via ferroptosis resistance dependent Nrf2 signaling pathway

BackgroundHepatocellular carcinoma (HCC) is a common malignancy with a poor prognosis. The recommended treatment of unresectable HCC involves targeted therapy, for example sorafenib, combined with immunotherapy. A recent article reported that sorafenib could induce ferroptosis escape in HCC. Brusatol is a novel Nrf2 inhibitor that takes effects in various diseases. In our study, we aimed to identify whether the addition of Brusatol to sorafenib could reverse ferroptosis escape in Huh7 cells. MethodsThe cultured Huh7 cells treated by sorafenib with or without Brusatol addition were harvested for ferroptotic phenotype experiments and ferroptosis-related markers such as GPX4 and SLC7A11 were detected. In vivo experiments were conducted to discover the effect of Brusatol in combination with sorafenib in liver tumor bearing mice. Mechanism signaling pathways were detected by RNA-sequencing. ResultsBrusatol alone could induce Huh7 cell death and sorafenib could moderately mediate Huh7 cell ferroptosis by paradoxically inhibiting GPX4. However, sorafenib simultaneously upregulates Nrf2 signaling in Huh7 cells fighting against ferroptosis to result in sorafenib resistance. The addition of Brusatol could potentiate ferroptosis in Huh7 cells through downregulating Nrf2 and the downstream HO-1 and NQO1, thus enhancing the efficacy of sorafenib, which could be reversed by ferrostatin-1 treatment. ConclusionIn conclusion, Brusatol improves the efficacy of sorafenib by inducing ferroptosis via hindering Nrf2 signaling activation in HCC.

Investigation of photothermal treatment with 532nm laser light on laryngeal cancer in an in vivo tumor model.

Laryngeal cancer is the second most common cancer in the upper aerodigestive tract,with its incidence increasing across all ages. The conventional treatments for laryngeal cancerinclude surgical procedure, radiation, and chemotherapy; however, these treatments can leadto various complications. Photothermal therapy (PTT) using laser light has been employed formcancer effective treatment because of its minimal invasion and short operation time. Thecurrent study aims to investigate the feasibility of 532 nm PTT on laryngeal cancer in an invivo tumor model.Ex vivo dosimetry evaluation was conducted to determine the laser irradiationconditions, and HEP-2 tumor bearing mice were used to demonstrate in vivo photothermaleffects. In addition, histology and western blot analysis were conducted to verify tumornecrosis and any changes in cancer-associated factors in the tumor tissues.The current in vivo results showed that PTT at 5 W for 40 s and 20 W for 10 s hadcomparable effects in terms of temperature increase and tumor removal. The 532 nm PTTsignificantly decreased the remaining tumor and downregulated the expression levels of MMP-9 and ERK.The current study demonstrated that the 532 nm PTT could be a feasible optionfor treatment of laryngeal tumor with high power delivery for a short exposure time. Furtherinvestigations will confirm the endoscopic application of the 532 nm PTT for the treatment ofintralaryngeal tissue prior to clinical translation.

Switchable PAMAM megamers for deep tumor penetration and enhanced cell uptake

Nanoparticles fabricated to deliver anticancer drugs are usually designed to present optimized tumor penetration and cell internalization. However, there are some barriers and difficulties with most current technologies. Herein, size and charge switchable polyamidoamine (PAMAM) megamers (SChPMs) were prepared for the delivery of doxorubicin (DOX). SChPMs were fabricated by connecting PAMAM dendrimers with pH-sensitive bonds and surface PEGylation. At pH 7.4, the size and surface charge of these nanocarriers were approximately 100 nm and + 0.75 mV, but at the acidic extracellular pH of tumor cells (pH 6.5), their size were reduced dramatically (15 nm) and their surface charge increased to +6.7 mV. Cell studies confirmed that alteration of the size and surface charge enhanced their penetration into multicellular spheroids and cell internalization. These megamers, in addition to delivering the drug to the deeper areas of the tumor, could powerfully overcome physiological resistance to anthracycline-based drugs. The nanocarrier revealed enhanced antitumoral activity in animal studies. Toxicology studies and histopathological assessments of vital tissues of 4 T1 tumor bearing mice indicated minimal tissue damage when DOX-loaded SChPMs (DSChPMs) were used. It can be concluded that the versatile and agile nanocarriers developed in this study could be considered for further investigations into their clinical application.

Abstract A073: Desmoglein-2 is a regulator of pancreatic ductal adenocarcinoma progression

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with a dismal 5-year survival rate of &amp;lt;13%. We have discovered that desmoglein-2 (DSG2), a cell surface protein, is highly expressed on PDAC cells. In silico data indicate that elevated levels of DSG2 is associated with PDAC progression and poor patient survival. Immunohistochemistry (IHC) analysis of a tissue microarray of 302 patient samples showed that DSG2 was expressed by &amp;gt;90% of PDAC patients. We hypothesized that DSG2 is an under-appreciated contributor to PDAC progression and that DSG2 can be targeted. The main aims of this study were to: 1) investigate the role of DSG2 in PDAC cell function in vitro, 2) investigate the molecular biology underpinning the role of DSG2 in PDAC, and 3) to use murine PDAC tumor models to evaluate the contribution of DSG2 to cancer progression in vivo. Our in vitro data showed that reducing the expression of DSG2 (via small interfering RNA (siRNA)) significantly decreased PDAC cell proliferation, migration, and invasion. Moreover, signaling analysis of PDAC cells with or without DSG2 (via siRNA) using a reverse-phase protein array and cytokine array revealed that DSG2 modulates pro-tumorigenic and pro-metastatic cellular pathways, through reducing the effects of certain proteins. Particular alterations were observed in pathways involving gene regulation (e.g., beta-catenin), migration (e.g., epidermal growth factor receptor and integrin-beta1), and cytokine release (e.g., serpine-1). Next, in an orthotopic xenograft mouse model, BxPC-3 PDAC cells with a stable knockdown of DSG2 (via short-hairpin RNA) were injected into NSG (NOD scid gamma) mice, where we observed a significant reduction in tumor burden and liver metastasis when compared to control mice. IHC staining of tumor sections revealed that the DSG2 knockdown tumors contained reduced levels of collagen, tumor vasculature and cancer associated fibroblasts. Thus, suggesting that DSG2 also has a modulatory effect on the tumor microenvironment (TME). A similar reduction in tumor burden, cancer progression and changes to the TME were observed when a neutralizing anti-DSG2 monoclonal antibody was administered intraperitoneally twice a week at 5mg/kg to BxPC-3 tumor bearing mice. Finally, to investigate the immune cell landscape, we used an orthotopic syngeneic mouse model, whereby Dsg2 was knocked out of KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) mouse PDAC cells via clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. This mouse model also showed that loss of Dsg2 resulted in a significant reduction in tumor burden, cancer cell metastasis and changes to the TME. In summary, we have identified a potential new target for the treatment of PDAC, namely DSG2, and future experiments are pursuing clinical opportunities to improve PDAC patient survival. Citation Format: Charlie B Ffrench, Kay K Myo Min, Mark DeNichilo, Michaelia P Cockshell, Emma L Dorward, Emma J Thompson, Michael Ortiz, Michael S Samuel, Savio George Barreto, Claudine S Bonder. Desmoglein-2 is a regulator of pancreatic ductal adenocarcinoma progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A073.

Abstract A059: Understanding the events that promote pancreatic cancer metastasis

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy due to overwhelming metastatic burden. PDAC cells migrate and invade by two distinct epithelial-to-mesenchymal transition phenotypes: collective (partial-EMT) or single-cell (complete-EMT). The mechanism(s) by which these invasive phenotypes intravasate is unknown. The Tumor Microenvironment of Metastasis (TMEM) doorway, which is formed by a tumor cell, macrophage and an endothelial cell in direct contact, causes opening of the vasculature in which tumor cells intravasate. Intravasation via TMEM doorway is inhibited by Tie2 blockade. Whether this occurs in PDAC and whether the EMT phenotype affects intravasation is unknown. We hypothesize that PDAC cells intravasate through TMEM-dependent mechanisms regardless of EMT phenotype. Tumor samples from murine models were stained for TMEM doorways using a triple immunohistochemistry (IHC) stain (tumor cells expressing the actin regulatory protein Mena, macrophages expressing CD68, and endothelial cells expressing CD31). TMEM doorway opening was assessed in murine models of PDAC treated with or without Tie-2 inhibitor by analyzing TMEM-mediated extravasation of high-molecular weight dextran (155kD-TMR dextran) by Intravital Imaging, IHC and immunofluorescence (IF) of serial slides and circulating tumor cells. An intravasation trans-endothelial migration assay was also performed to assess the intravasation event by TMEM doorway in vitro. A student t-test was used for the analyses. TMEM doorways were observed in all PDAC murine models. For the first time in PDAC, focal extravasation of dextran events followed by a tumor cell intravasation was visualized by intravital imaging. PDAC tumor bearing mice treated with Tie2 inhibitor had a 2-fold decrease in the number of circulating tumor cells compared to control (958 ± 499.8 vs 534 ± 479.9 cells/mL blood, p=0.0257). The intravasation trans-endothelial migration assay showed that C-EMT cells have a higher migration rate than P-EMT cells (17.8 ± 5.53 vs 10.94 ± 3.84 migrating cells p=0.01), and this ratio increases with the presence of macrophages (23.75 ± 5 vs 15.54 ± 3.09 migrating cells p=0.005). The Tie-2 inhibition decreases the number of C-EMT and P-EMT migrating cells (7.69 ± 1.3 vs 3.67 ± 1.2 migrating cells p=0.01). Orthotopic tumors from C-EMT phenotype grow at a faster rate than the P-EMT (0.49mg ± 0.09 vs 0.12mg ± 0.05 p&amp;lt;0.05). Tumors from C-EMT and P-EMT cells will be analyzed for TMEM doorway score, TMEM activity and stemness signatures around TMEM doorway to determine if TMEM doorway is a niche for stemness in PDAC. TMEM doorways are present in PDAC mouse model. Inhibition of Tie2 leads to decreased TMEM doorway function and circulating tumor cells in PDAC. In addition, regarding the EMT phenotype, TMEM doorways are functional mechanisms of intravasation in vitro. This is a novel mechanism in PDAC and suggests a promising therapeutic target for decreasing intravasation and potential metastasis in PDAC. Citation Format: Erika Pereira Zambalde, Lina Ariyan, Francisco Puerta-Martinez, Yuqin Zhu, John Condeelis, Ben Stanger, John McAuliffe. Understanding the events that promote pancreatic cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A059.

Validation of the C-X-C chemokine receptor 3 (CXCR3) as a target for PET imaging of T cell activation

PurposeCXCR3 is expressed on activated T cells and plays a crucial role in T-cell recruitment to the tumor microenvironment (TME) during cell-based and immune checkpoint inhibitor (ICI) immunotherapy. This study utilized a 64Cu-labeled NOTA-α-CXCR3 antibody to assess CXCR3 expression in the TME and validate it as a potential T cell activation biomarker in vivo.ProceduresCXCR3+ cells infiltrating MC38 tumors (B57BL/6 mice, untreated and treated with αPD-1/αCTLA-4 ICI) were quantified using fluorescence microscopy and flow cytometry. A commercial anti-mouse CXCR3 antibody (α-CXCR3) was site-specifically conjugated with 2,2,2-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid (NOTA) and radiolabeled with 64Cu. Saturation binding of [64Cu]Cu-NOTA-α-CXCR3 was investigated using CHO cells stably transfected with murine CXCR3. Biodistribution and PET imaging studies both at baseline and after 1 to 3 cycles of ICI, respectively, were carried out using different molar activities (10 GBq/µmol to 300 GBq/µmol) of [64Cu]Cu-NOTA-α-CXCR3.ResultsFlow cytometry analysis at baseline confirmed the presence of CXCR3 + T-cells in MC38 tumors, which was significantly increased at day five after ICI (treated 33.8 ± 17.4 vs. control 8.8 ± 6.2 CD3+CXCR3+ cells/mg). These results were qualitatively and quantitatively confirmed by immunofluorescence of tumor cryoslices. In vivo PET imaging of MC38 tumor bearing mice before, during and after ICI using [64Cu]Cu-NOTA-α-CXCR3 (Kd = 3.3 nM) revealed a strong dependence of CXCR3-specificity of tracer accumulation in secondary lymphoid organs on molar activity. At 300 GBq/µmol (1.5 µg of antibody/mouse), a specific signal was observed in lymph nodes (6.33 ± 1.25 control vs. 3.95 ± 1.23%IA/g blocking) and the spleen (6.04 ± 1.02 control vs. 3.84 ± 0.79%IA/g blocking) at 48 h p.i. Spleen-to-liver ratios indicated a time dependent systemic immune response showing a steady increase from 1.08 ± 0.19 (untreated control) to 1.54 ± 0.14 (three ICI cycles).ConclusionsThis study demonstrates the feasibility of in vivo imaging of CXCR3 upregulation under immunotherapy using antibodies. However, high molar activities and low antibody doses are essential for sensitive detection in lymph nodes and spleen. Detecting therapy-induced changes in CXCR3+ T cell numbers in tumors was challenging due to secondary antibody-related effects. Nonetheless, CXCR3 remains a promising target for imaging T cell activation, with anticipated improvements in sensitivity using alternative tracers with high affinities and favorable pharmacokinetics.

Hypoxia-induced complement component 3 promotes aggressive tumor growth in the glioblastoma microenvironment.

Glioblastoma (GBM) is the most aggressive form of glioma with a high rate of relapse despite intensive treatment. Tumor recurrence is tightly linked to radio-resistance, which in turn is associated with hypoxia. Here, we discovered a strong link between hypoxia and local complement signaling using publicly available bulk, single-cell, and spatially resolved transcriptomic data from patients with GBM. Complement component 3 (C3) and the receptor C3AR1 were both associated with aggressive disease and shorter survival in human glioma. In a genetically engineered mouse model of GBM, we found C3 specifically in hypoxic tumor areas. In vitro, we found an oxygen level-dependent increase in C3 and C3AR1 expression in response to hypoxia in several GBM and stromal cell types. C3a induced M2 polarization of cultured microglia and macrophages in a C3aR-dependent fashion. Targeting C3aR using the antagonist SB290157 prolonged survival of glioma-bearing mice both alone and in combination with radiotherapy while reducing the number of M2-polarized macrophages. Our findings establish a strong link between hypoxia and complement pathways in GBM and support a role of hypoxia-induced C3a/C3aR signaling as a contributor to glioma aggressiveness by regulating macrophage polarization.

Spatiotemporal coordination of antigen presentation and co-stimulatory signal for enhanced anti-tumor vaccination

Controlled-release systems enhance anti-tumor effects by leveraging local antigen persistence for antigen-presenting cells (APCs) recruitment and T cell engagement. However, constant antigen presentation alone tends to induce dysfunction in tumor-specific CD8+ T cells, neglecting the synergistic effects of co-stimulatory signal. To address this, we developed a soft particle-stabilized emulsion (SPE) to deliver lipopeptides with controlled release profiles by adjusting their hydrophobic chain lengths: C6-SPE (fast release), C10-SPE (medium release), and C16-SPE (slow release). Following administration, C6-SPE release antigen rapidly, inducing early antigen presentation, whereas C16-SPE's slow-release delays antigen presentation. Both scenarios missed the critical window for coordinating with the expression of CD86, leading to either T cell apoptosis or suboptimal activation. In contrast, C10-SPE achieved a spatiotemporally synergetic effect of the MHC-I-peptide complex and co-stimulatory signal (CD86), leading to effective dendritic cell (DC) activation, enhanced T cell activation, and tumor regression in EG7-OVA bearing mice. Additionally, co-delivery of cytosine-phosphate-guanine (CpG) with SPE provided a sustained expression of the CD86 window for DC activation, improving the immune response and producing robust anti-tumor effects with C6-SPE comparable to C10-SPE. These findings highlight that synchronizing the spatiotemporal dynamics of antigen presentation and APC activation may confer an optimal strategy for enhanced vaccinations.

Whey protein and flaxseed gum co-encapsulated fucoxanthin promoted tumor cells apoptosis based on MAPK-PI3K/Akt regulation on Huh-7 cell xenografted nude mice

Fucoxanthin (FX), a non-provitamin-A carotenoid, is a well-known major xanthophyll contained in edible brown algae. The nanoencapsulation of FX was motivated due to its multiple activities. Here, nano-encapsulated-FX (nano-FX) was prepared according to our early method by using whey protein and flaxseed gum as the biomacromolecule carrier material, then in vivo antitumor effect and mechanism of nano-FX on xenograft mice were investigated. Thirty 4-week-old male BALB/c nude mice were fed adaptively for 7 days to establish xenograft tumor model with Huh-7 cells. The tumor-bearing mice consumed nano-FX (50, 25, and 12.5 mg kg−1) and doxorubicin hydrochloride (DOX, 1 mg kg−1) or did not consume (Control) for 21 days, n = 6. The tumor inhibition rates of nano-FX were as high as 54.67 ± 1.04 %. Nano-FX intervention promoted apoptosis and induced hyperchromatic pyknosis and focal necrosis in tumor tissue by down-regulating the expression of p-JNK, p-ERK, PI3Kp85α, p-AKT, p-p38MAPK, Bcl-2, CyclinD1 and Ki-67, while up-regulating the expression of cleaved caspase-3 and Bax. Nano-FX inhibited tumor growth and protected liver function of tumor bearing mice in a dose-dependent manner, up-regulate the level of apoptosis-related proteins, inhibit the MAPK-PI3K/Akt pathways, and promote tumor cell apoptosis.

Revealing the protective role of mitophagy for liver injury via a promising dual-channel activated fluorescent probe

As a highly active organelle in the regulation of hepatic cellular redox, the dysfunction of mitochondrial is tightly associated with both acute and chronic liver diseases. Mitophagy, the major pathway to maintenance of mitochondrial quality plays a vital role in the regulation of liver homeostasis by emerging evidence. Real time monitoring two or more mitochondrial microenvironment parameters alterations during mitophagy is more favorable for better elucidating the mitophagy process and its mechanism of action for liver disease. Here, we proposed a dual responsive probe named SP-DCN through effective crosslinking the fragments of spiropyrane and dicyanoisophorone for simultaneously monitoring of pH and viscosity. The mitophagy induced by different external stimulations all lead to the same changing trends of intracellular fluorescence signals in the two imaging channels. The enhancement of fluorescence ratio value in two channels were obtained in the tumor site and liver site of tumor bearing and liver injury mice, respectively. It was noted that the fluorescence ratio value could be served as a characteristic parameter to evaluate the intracellular mitophagy level. More importantly, the proposed dual-channel activated imaging method was successfully applied to confirm the protective effect of mitophagy against drug induced liver injury. This approach holds promise for further analysis the regulatory mechanisms of mitophagy in liver, and contributes to identify promising targets for the future treatment of liver diseases.