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JCI Insight Dec 2023IL-12 is a potent cytokine that can promote innate and adaptive anticancer immunity, but its clinical development has been limited by toxicity when delivered...
IL-12 is a potent cytokine that can promote innate and adaptive anticancer immunity, but its clinical development has been limited by toxicity when delivered systemically. Intratumoral (i.t.) administration can expand the therapeutic window of IL-12 and other cytokines but is in turn limited by rapid drug clearance from the tumor, which reduces efficacy, necessitates frequent administration, and increases systemic accumulation. To address these limitations, we developed an anchored IL-12 designated ANK-101, composed of an engineered IL-12 variant that forms a stable complex with the FDA-approved vaccine adjuvant aluminum hydroxide (Alhydrogel). Following i.t. administration of murine ANK-101 (mANK-101) in early intervention syngeneic mouse tumors, the complex formed a depot that was locally retained for weeks as measured by IVIS or SPECT/CT imaging, while unanchored protein injected i.t. was cleared within hours. One or 2 i.t. injections of mANK-101 induced single-agent antitumor activity across a diverse range of syngeneic tumors, including models resistant to checkpoint blockade at doses where unanchored IL-12 had no efficacy. Local treatment with mANK-101 further induced regressions of noninjected lesions, especially when combined with systemic checkpoint blockade. Antitumor activity was associated with remodeling of the tumor microenvironment, including prolonged IFN-γ and chemokine expression, recruitment and activation of T and NK cells, M1 myeloid cell skewing, and increased antigen processing and presentation. Subcutaneous administration of ANK-101 in cynomolgus macaques was well tolerated. Together, these data demonstrate that ANK-101 has an enhanced efficacy and safety profile and warrants future clinical development.
Topics: Mice; Animals; Interleukin-12; Aluminum Hydroxide; Tumor Microenvironment; Cytokines; Neoplasms
PubMed: 38063196
DOI: 10.1172/jci.insight.168224 -
Journal of Visualized Experiments : JoVE Oct 2022Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, immune cells,...
Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, immune cells, vessels, nerves, and extracellular matrix components. Over the years, different mouse models of PDAC have been developed to address the challenges posed by its progression, metastatic potential, and phenotypic heterogeneity. Immunocompetent mouse orthotopic allografts of PDAC have shown good promise owing to their fast and reproducible tumor progression in comparison to genetically engineered mouse models. Moreover, combined with their ability to mimic the biological features observed in autochthonous PDAC, cell line-based orthotopic allograft mouse models enable large-scale in vivo experiments. Thus, these models are widely used in preclinical studies for rapid genotype-phenotype and drug-response analyses. The aim of this protocol is to provide a reproducible and robust approach to successfully inject primary mouse PDAC cell cultures into the pancreas of syngeneic recipient mice. In addition to the technical details, important information is given that must be considered before performing these experiments.
Topics: Mice; Animals; Pancreatic Neoplasms; Carcinoma, Pancreatic Ductal; Pancreas; Tumor Microenvironment; Disease Models, Animal; Allografts; Cell Line, Tumor
PubMed: 36282716
DOI: 10.3791/64253 -
Tissue Engineering. Part A Nov 2020The immunogenicity of the extracellular matrix (ECM) from genetically similar (syngeneic) and dissimilar (allogeneic and xenogeneic) species has puzzled the scientific...
The immunogenicity of the extracellular matrix (ECM) from genetically similar (syngeneic) and dissimilar (allogeneic and xenogeneic) species has puzzled the scientific community for many years. After implantation, the literature describes an absorption of ECM material since it is biodegradable. However, no clear insight really exists to substantiate how the underlying immune and biological responses result in absorption of ECM materials. In this context, it is important to characterize infiltrating cells and identify dominant cell populations in the infiltrate. We have studied the immune response in mice after implantation of decellularized (DC) cardiac scaffolds derived from pig and mouse. The polymorphism of the infiltrate into the implanted material signifies the importance of the adaptive immune response that is distinct for xenoimplants and alloimplants. Matrix resorption takes place mainly through phagocytic cells such as mast cells, dendritic cells, and macrophages. Histochemical observations show that innate CD8 T cells develop immune tolerance, whereas proteomic analysis predicts the different T cell progenies for alloscaffolds and xenoscaffolds. The amalgamation of graft tolerance and involvement of both B and T cell populations in the vicinity of the graft could be decisive in wound remodeling and survival of the graft. This challenging area presents potential targets for the development of immune-privileged biomaterials, immune tolerant cells, and therapeutic agents in the future. Impact statement In this study, we have characterized the allogeneic and xenogeneic immune responses for decellularized (DC) cardiac scaffolds. We postulate that although the T cells are important players for immune tolerance of DC graft, the mechanism of their differentiation inside the host is donor specific. In this study, we have reported the distinct immune responses for syngeneic DC scaffolds than allogeneic and xenogeneic scaffolds. This distinct response provides the bases for the different immune responses reported for DC homografts in the literature. This study can provide the greater insight for modification of postimplant strategies to achieve host acceptance of donor extracellular matrix scaffolds.
Topics: Animals; Biocompatible Materials; CD8-Positive T-Lymphocytes; Extracellular Matrix; Heart; Heterografts; Immune Tolerance; Mice; Proteomics; Swine
PubMed: 32484039
DOI: 10.1089/ten.TEA.2020.0055 -
Molecular Pharmaceutics May 2023Tumor-associated macrophages (TAMs) are large phagocytic cells that play numerous roles in cancer biology and are an important component of the relationship between...
Tumor-associated macrophages (TAMs) are large phagocytic cells that play numerous roles in cancer biology and are an important component of the relationship between immune system response and tumor progression. The peptide, RP832c, targets the Mannose Receptor (CD206) expressed on M2-like macrophages and is cross-reactive to both human and murine CD206. Additionally, it exhibits therapeutic properties through its ability to shift the population of TAMs from an M2-like (protumor) toward an M1-like phenotype (antitumor) and has demonstrated promise in inhibiting tumor resistance in PD-L1 unresponsive melanoma murine models. In addition, it has shown inhibition in bleomycin-induced pulmonary fibrosis through interactions with CD206 macrophages. Our work aims to develop a novel CD206 positron emission tomography (PET) imaging probe based on RP832c ( = 5.64 μM) as a direct, noninvasive method for the assessment of TAMs in mouse models of cancer. We adapted RP832c to incorporate the chelator DOTA to allow for radiolabeling with the PET isotope Ga ( = 68 min; ß = 89%). In vitro stability studies were conducted in mouse serum up to 3 h. The in vitro binding characteristics of [Ga]RP832c to CD206 were determined by a protein plate binding assay and Surface Plasmon Resonance (SPR). PET imaging and biodistribution studies were conducted in syngeneic tumor models. Stability studies in mouse serum demonstrated that Ga remained complexed up to 3 h (less than 1% free Ga). Binding affinity studies demonstrated high binding of [Ga]RP832c to mouse CD206 protein and that the binding of the tracer was able to be blocked significantly when incubated with a blocking solution of native RP832c. PET imaging and biodistribution studies in syngeneic tumor models demonstrated uptake in tumor and CD206 expressing organs of [Ga]RP832c. A significant correlation was found between the percentage of CD206 present in each tumor imaged with [Ga]RP832c and PET imaging mean standardized uptake values in a CT26 mouse model of cancer. The data shows that [Ga]RP832c represents a promising candidate for macrophage imaging in cancer and other diseases.
Topics: Animals; Humans; Mice; Cell Line, Tumor; Gallium Radioisotopes; Macrophages; Neoplasms; Peptides; Positron-Emission Tomography; Tissue Distribution; Mannose Receptor
PubMed: 37014648
DOI: 10.1021/acs.molpharmaceut.2c00977 -
Scientific Reports Feb 2022Cancers are immunologically heterogeneous. A range of immunotherapies target abnormal tumor immunity via different mechanisms of actions (MOAs), particularly various...
Cancers are immunologically heterogeneous. A range of immunotherapies target abnormal tumor immunity via different mechanisms of actions (MOAs), particularly various tumor-infiltrate leukocytes (TILs). We modeled loss of function (LOF) in four common anti-PD-1 antibody-responsive syngeneic tumors, MC38, Hepa1-6, CT-26 and EMT-6, by systematical depleting a series of TIL lineages to explore the mechanisms of tumor immunity and treatment. CD8-T-cells, CD4-T-cells, T, NK cells and macrophages were individually depleted through either direct administration of anti-marker antibodies/reagents or using DTR (diphtheria toxin receptor) knock-in mice, for some syngeneic tumors, where specific subsets were depleted following diphtheria toxin (DT) administration. These LOF experiments revealed distinctive intrinsic tumor immunity and thus different MOAs in their responses to anti-PD-1 antibody among different syngeneic tumors. Specifically, the intrinsic tumor immunity and the associated anti-PD-1 MOA were predominately driven by CD8 cytotoxic TILs (CTL) in all syngeneic tumors, excluding Hepa1-6 where CD4 T TILs played a key role. TIL-T also played a critical role in supporting tumor growth in all four syngeneic models as well as M-macrophages. Pathway analysis using pharmacodynamic readouts of immuno-genomics and proteomics on MC38 and Hepa1-6 also revealed defined, but distinctive, immune pathways of activation and suppression between the two, closely associated with the efficacy and consistent with TIL-pharmacodynamic readouts. Understanding tumor immune-pathogenesis and treatment MOAs in the different syngeneic animal models, not only assists the selection of the right model for evaluating new immunotherapy of a given MOA, but also can potentially help to understand the potential disease mechanisms and strategize optimal immune-therapies in patients.
Topics: Animals; Antineoplastic Agents; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Humans; Immunotherapy; Lymphocytes, Tumor-Infiltrating; Mice; T-Lymphocytes, Regulatory; Tumor Microenvironment
PubMed: 35228603
DOI: 10.1038/s41598-022-07153-z -
Bio-protocol Sep 2020Endometriosis is a common gynecological disease characterized by the presence of endometrial tissue outside the uterine cavity. It is frequently associated with pain,...
Endometriosis is a common gynecological disease characterized by the presence of endometrial tissue outside the uterine cavity. It is frequently associated with pain, infertility and a reduced quality of life, and it lacks adequate treatment. Several rodent models of endometriosis have been developed through heterologous and homologous transplantation of endometrial tissue into the abdominal compartment. Here we describe a surgical procedure to generate a syngeneic model of endometriosis in immunocompetent mice with intact uterine and ovarian tissues. In this model, four uterine fragments from a donor mouse at diestrus are sutured to the abdominal wall of a recipient mouse. One month after surgeries, endometrial implants develop into cysts with glandular epithelium and stroma, mimicking the endometriotic lesions observed in women with endometriosis. Therefore, this mouse model provides a valuable tool to study the pathophysiology of endometriosis and the efficacy of potential treatments.
PubMed: 33659421
DOI: 10.21769/BioProtoc.3763 -
Cancer Growth and Metastasis 2015Many common cancers have a propensity to metastasize to bone. Although malignancies often go undetected in their native tissues, bone metastases produce excruciating... (Review)
Review
Many common cancers have a propensity to metastasize to bone. Although malignancies often go undetected in their native tissues, bone metastases produce excruciating pain that severely compromises patient quality of life. Cancer-induced bone pain (CIBP) is poorly managed with existing medications, and its multifaceted etiology remains to be fully elucidated. Novel analgesic targets arise as more is learned about this complex and distinct pain state. Over the past two decades, multiple animal models have been developed to study CIBP's unique pathology and identify therapeutic targets. Here, we review animal models of CIBP and the mechanistic insights gained as these models evolve. Findings from immunocompromised and immunocompetent host systems are discussed separately to highlight the effect of model choice on outcome. Gaining an understanding of the unique neuromolecular profile of cancer pain through the use of appropriate animal models will aid in the development of more effective therapeutics for CIBP.
PubMed: 26339191
DOI: 10.4137/CGM.S21215 -
Faithful preclinical mouse models for better translation to bedside in the field of immuno-oncology.International Journal of Clinical... May 2020The success of immunotherapy using immune checkpoint inhibitors has changed the practice of cancer treatment tremendously. However, there are still many clinical... (Review)
Review
The success of immunotherapy using immune checkpoint inhibitors has changed the practice of cancer treatment tremendously. However, there are still many clinical challenges, such as drug resistance, predictive biomarker development, exploration of combination therapies, and prediction of immune-related adverse events in preclinical settings. To overcome these problems, it is essential to establish faithful preclinical mouse models that recapitulate the clinical features, molecular genetics, biological heterogeneity, and immune microenvironment of human cancers. Here we review the advantages and disadvantages of current preclinical mouse models, including syngeneic murine tumor cell lines, autochthonous tumor models, cancer cell line-derived xenografts, patient-derived-xenografts, and various kinds of immunologically humanized mice. We discuss how these models should be characterized and applied in preclinical settings, and how we should prepare preclinical studies for successful translation from bench to bedside.
Topics: Animals; Cell Line, Tumor; Humans; Immunotherapy; Mice; Neoplasms, Experimental; Translational Research, Biomedical; Vertebrates; Xenograft Model Antitumor Assays
PubMed: 31407168
DOI: 10.1007/s10147-019-01520-z -
Breast Cancer Research : BCR Aug 2021The heterogeneity of the breast tumor microenvironment (TME) may contribute to the lack of durable responses to immune checkpoint blockade (ICB); however, mouse models... (Comparative Study)
Comparative Study
BACKGROUND
The heterogeneity of the breast tumor microenvironment (TME) may contribute to the lack of durable responses to immune checkpoint blockade (ICB); however, mouse models to test this are currently lacking. Proper selection and use of preclinical models are necessary for rigorous, preclinical studies to rapidly move laboratory findings into the clinic.
METHODS
Three versions of a common syngeneic model derived from the MMTV-PyMT autochthonous model were generated by inoculating 1E6, 1E5, or 1E4 cells derived from the MMTV-PyMT mouse into wildtype recipient mice. To elucidate how tumor latency and TME heterogeneity contribute to ICB resistance, comprehensive characterization of the TME using quantitative flow-cytometry and RNA expression analysis (NanoString) was performed. Subsequently, response to ICB was tested. These procedures were repeated using the EMT6 breast cancer model.
RESULTS
The 3 syngeneic versions of the MMTV-PyMT model had vastly different TMEs that correlated to ICB response. The number of cells used to generate syngeneic tumors significantly influenced tumor latency, infiltrating leukocyte populations, and response to ICB. These results were confirmed using the EMT6 breast cancer model. Compared to the MMTV-PyMT autochthonous model, all 3 MMTV-PyMT syngeneic models had significantly more tumor-infiltrating lymphocytes (TILs; CD3, CD4, and CD8) and higher proportions of PD-L1-positive myeloid cells, whereas the MMTV-PyMT autochthonous model had the highest frequency of myeloid cells out of total leukocytes. Increased TILs correlated with response to anti-PD-L1 and anti-CTLA-4 therapy, but PD-L1expression on tumor cells or PD-1 expression of T cells did not.
CONCLUSIONS
These studies reveal that tumor cell number correlates with tumor latency, TME, and response to ICB. ICB-sensitive and resistant syngeneic breast cancer models were identified, in which the 1E4 syngeneic model was most resistant to ICB. Given the lack of benefit from ICB in breast cancer, identifying robust murine models presented here provides the opportunity to further interrogate the TME for breast cancer treatment and provide novel insights into therapeutic combinations to overcome ICB resistance.
Topics: Animals; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Immune Checkpoint Inhibitors; Immunotherapy; Lymphocytes, Tumor-Infiltrating; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Myeloid Cells; Neoplasm Transplantation; T-Lymphocytes; Transcriptome; Transplantation, Isogeneic; Tumor Microenvironment
PubMed: 34353349
DOI: 10.1186/s13058-021-01448-1 -
Methods in Molecular Biology (Clifton,... 2023Cancer-associated fibroblasts (CAFs) are vital within the tumor ecosystem, regulating tumor growth, dissemination, and response to therapy through crosstalk with tumor...
Cancer-associated fibroblasts (CAFs) are vital within the tumor ecosystem, regulating tumor growth, dissemination, and response to therapy through crosstalk with tumor cells, infiltrating immune and vascular cells, as well as components of the extracellular matrix (ECM). CAFs have thus emerged as potential therapeutic targets to complement cancer cell-targeted therapies. To study CAF-tumor cell crosstalk ex vivo, robust isolation methods of primary CAFs are required. Here, we present protocols to isolate, expand, and culture two types of fibroblasts: (1) healthy murine mammary gland fibroblasts, a key source of the CAF population in breast tumor models and (2) CAFs derived from syngeneic murine breast tumors. Isolated mammary fibroblasts and CAFs are suitable for use in a variety of downstream cellular and molecular experiments. We expect these methods to be useful to scientists studying the properties of fibroblasts and CAFs and the interaction between CAFs and the various components of the tumor microenvironment (TME).
Topics: Mice; Animals; Ecosystem; Fibroblasts; Cancer-Associated Fibroblasts; Mammary Neoplasms, Animal; Tumor Microenvironment
PubMed: 36587126
DOI: 10.1007/978-1-0716-2914-7_12