Human Mouse Research Articles

IGSF8 is an innate immune checkpoint and cancer immunotherapy target

Antigen presentation defects in tumors are prevalent mechanisms of adaptive immune evasion and resistance to cancer immunotherapy, whereas how tumors evade innate immunity is less clear. Using CRISPR screens, we discovered that IGSF8 expressed on tumors suppresses NK cell function by interacting with human KIR3DL2 and mouse Klra9 receptors on NK cells. IGSF8 is normally expressed in neuronal tissues and is not required for cell survival invitro or invivo. It is overexpressed and associated with low antigen presentation, low immune infiltration, and worse clinical outcomes in many tumors. An antibody that blocks IGSF8-NK receptor interaction enhances NK cell killing of malignant cells invitro and upregulates antigen presentation, NK cell-mediated cytotoxicity, and Tcell signaling invivo. In syngeneic tumor models, anti-IGSF8 alone, or in combination with anti-PD1, inhibits tumor growth. Our results indicate that IGSF8 is an innate immune checkpoint that could be exploited as a therapeutic target.

Mouse models of CLL: In vivo modeling of disease initiation, progression, and transformation to Richter transformation

Chronic lymphocytic leukemia (CLL) is a highly complex disease characterized by the proliferation of CD5+ B cells in lymphoid tissues. Current modern treatments have brought significant clinical benefits to CLL patients. However, there are still unmet needs. Patients relapse on Bruton's tyrosine kinase inhibitors and BCL2 inhibitors and often develop more aggressive diseases including Richter transformation (RT), an incurable complication of up to ∼10% patients. This evidence underscores the need for improved immunotherapies, combination treatment strategies, and predictive biomarkers. A mouse model that can recapitulate human CLL disease and certain components of the tumor immune microenvironment represents a promising preclinical tool for such purposes. In this review, we provide an overview of CRISPR-engineered and xenograft mouse models utilizing either cell lines, or primary CLL cells suitable for studies of key events driving the disease onset, progression and transformation of CLL. We also review how CRISPR/Cas9 established mouse models carrying loss-of-function lesions allow one to study key mutations driving disease progression. Finally, we discuss how next generation humanized mice might improve to generation of faithful xenograft mouse models of human CLL.

Experimental Animal Research in Vaccine Studies during the Pandemic Process

The pandemic that emerged in Wuhan, China, in 2019, once again revealed the role of basic sciences and experimental animal research in the world. This situation, also called COVID-19, has spread rapidly all over the world in a short period of 3 months. It has been revealed that vaccination is more important than treatment in reducing mass deaths during the fight against the pandemic. The fact that RNA viruses constantly change their genetic properties was also a major challenge in finding an effective vaccine. However, increasing progress in basic sciences (especially mRNA technology) and the use of humanized mice have contributed greatly to reducing mass deaths. The fact that the Sars-Cov-2 virus does not cause infection in conventional mice and that it can create an infection model in mutant mice to which human lung epithelial genes are transferred has given great impetus to vaccine and new drug treatment studies. The first vaccine model was developed using these mice in vaccine studies. Nearly 176 vaccine types have been developed in the Covid-19 pandemic. These vaccines; viron vaccines are nucleic acid-based (mRNA), viral vector and protein-based vaccines. The mutant mice used in the first step of these vaccines created the disease model almost perfectly and ensured that the results in the target species (Human) progressed with fewer errors. Since the disease occurs in mice carrying human lung epithelium very close to the target species, they have played a major role in testing the protective effects of vaccines. The vaccines prepared towards the end of 2020 were made ready for use. Vaccine production has been carried out at an almost record speed in pandemics in world history. Although vaccines may have different effects and protection levels, the role of vaccines in the pandemic is undeniable. More than 2.5 billion doses of COVID-19 vaccines have been administered worldwide, and approximately 35 million doses continue to be administered every day. Experimental animal research, genetic research and gene transfer that started in the 1970s were used to obtain vaccines that were prepared in a short time and made available to humanity. technologies and mutant mouse technology play a huge role. Although the pandemic process is still not over, it is expected to ease and continue to have effects similar to seasonal flu. In this process, humanity has gained important experience in acting together and rapid vaccine development. The use of experimental animals is of great importance in these processes.

Preclinical evaluation of the SARS-CoV-2 Mpro inhibitor RAY1216 shows improved pharmacokinetics compared with nirmatrelvir

Although vaccines are available for SARS-CoV-2, antiviral drugs such as nirmatrelvir are still needed, particularly for individuals in whom vaccines are less effective, such as the immunocompromised, to prevent severe COVID-19. Here we report an α-ketoamide-based peptidomimetic inhibitor of the SARS-CoV-2 main protease (Mpro), designated RAY1216. Enzyme inhibition kinetic analysis shows that RAY1216 has an inhibition constant of 8.4 nM and suggests that it dissociates about 12 times slower from Mpro compared with nirmatrelvir. The crystal structure of the SARS-CoV-2 Mpro:RAY1216 complex shows that RAY1216 covalently binds to the catalytic Cys145 through the α-ketoamide group. In vitro and using human ACE2 transgenic mouse models, RAY1216 shows antiviral activities against SARS-CoV-2 variants comparable to those of nirmatrelvir. It also shows improved pharmacokinetics in mice and rats, suggesting that RAY1216 could be used without ritonavir, which is co-administered with nirmatrelvir. RAY1216 has been approved as a single-component drug named ‘leritrelvir’ for COVID-19 treatment in China.

SpatialcoGCN: deconvolution and spatial information-aware simulation of spatial transcriptomics data via deep graph co-embedding.

Spatial transcriptomics (ST) data have emerged as a pivotal approach to comprehending the function and interplay of cells within intricate tissues. Nonetheless, analyses of ST data are restricted by the low spatial resolution and limited number of ribonucleic acid transcripts that can be detected with several popular ST techniques. In this study, we propose that both of the above issues can be significantly improved by introducing a deep graph co-embedding framework. First, we establish a self-supervised, co-graph convolution network-based deep learning model termed SpatialcoGCN, which leverages single-cell data to deconvolve the cell mixtures in spatial data. Evaluations of SpatialcoGCN on a series of simulated ST data and real ST datasets from human ductal carcinoma in situ, developing human heart and mouse brain suggest that SpatialcoGCN could outperform other state-of-the-art cell type deconvolution methods in estimating per-spot cell composition. Moreover, with competitive accuracy, SpatialcoGCN could also recover the spatial distribution of transcripts that are not detected by raw ST data. With a similar co-embedding framework, we further established a spatial information-aware ST data simulation method, SpatialcoGCN-Sim. SpatialcoGCN-Sim could generate simulated ST data with high similarity to real datasets. Together, our approaches provide efficient tools for studying the spatial organization of heterogeneous cells within complex tissues.

Rapamycin increases leukemia cell sensitivity to chemotherapy by regulating mTORC1 pathway-mediated apoptosis and autophagy.

This study investigated the effect of rapamycin alone and in combination with chemotherapy (doxorubicin and cytarabine) on AML. Human acute monocytic leukemia cell line SHI-1 and NPG AML model mice created by intravenous injection of SHI-1 cell were treated with rapamycin, chemotherapy, or rapamycin plus chemotherapy. Analysis by cell counting kit-8, western blot, flow cytometry, and immunohistochemistry was performed, and results suggested that both rapamycin and chemotherapy inhibited proliferation of SHI-1 cells both in vitro and in vivo, suppressed neoplasm growth in vivo, and promoted survival of NPG AML mice. The antitumor effect of rapamycin plus chemotherapy was better than that of rapamycin alone and chemotherapy alone. In addition, western blot results demonstrated that rapamycin inhibited the phosphorylation of mTOR downstream targets 4EBP1 and S6K1 in SHI-1 cells, and increased the pro-apoptosis-related protein Bax and autophagy-associated proteins Beclin-1, LC3B-II, and ATG5 while reducing the anti-apoptosis-related protein Bcl-2. In conclusion, the results of this study indicate that rapamycin acts synergistically with doxorubicin and cytarabine in AML treatment, and its underlying mechanism might be associated with mTORC1 pathway-mediated apoptosis and autophagy.

Piezo1 channels restrain ILC2s and regulate the development of airway hyperreactivity.

Mechanosensitive ion channels sense force and pressure in immune cells to drive the inflammatory response in highly mechanical organs. Here, we report that Piezo1 channels repress group 2 innate lymphoid cell (ILC2)-driven type 2 inflammation in the lungs. Piezo1 is induced on lung ILC2s upon activation, as genetic ablation of Piezo1 in ILC2s increases their function and exacerbates the development of airway hyperreactivity (AHR). Conversely, Piezo1 agonist Yoda1 reduces ILC2-driven lung inflammation. Mechanistically, Yoda1 inhibits ILC2 cytokine secretion and proliferation in a KLF2-dependent manner, as we found that Piezo1 engagement reduces ILC2 oxidative metabolism. Consequently, in vivo Yoda1 treatment reduces the development of AHR in experimental models of ILC2-driven allergic asthma. Human-circulating ILC2s express and induce Piezo1 upon activation, as Yoda1 treatment of humanized mice reduces human ILC2-driven AHR. Our studies define Piezo1 as a critical regulator of ILC2s, and we propose the potential of Piezo1 activation as a novel therapeutic approach for the treatment of ILC2-driven allergic asthma.

Abstract 5345: Development and validation of humanized HLA mouse model platforms for preclinical evaluation of novel peptide vaccines

Abstract The human leukocyte antigen (HLA) class I major histocompatibility complex (MHC) plays an integral role in immune surveillance by presenting intracellular antigen peptides on the cell surface, enabling recognition by cytotoxic T cells and subsequent elimination. Harnessing this intrinsic process for cancer immunotherapy is a promising therapeutic strategy; thus, developing preclinical models to evaluate the efficacy of novel peptide vaccines in vivo and ex vivo is critical. Previously, we developed a humanized MHC I mouse model (B-HLA-A2.1 mice), in which we successfully demonstrated efficacy of peptide vaccines as assessed by splenocyte IFN-γ production measured via ELISpot. Here, we provide evidence that B-HLA-A2.1 mice can also mount immune responses to NY-ESO-1 and WT1-Db126 peptides using the same assay. Additionally, immunization with WT1-Db126 at 7, 14, and 21 days prior to tumor inoculation with WT1-expressing MC38 colon cancer cells significantly reduced tumor volume. Additionally, we have generated two new humanized HLA models: B-HLA-A11.1 mice and B-HLA-A24.2 mice. Flow cytometric data demonstrate successful expression of the human HLAs on the cell surface of splenocytes isolated from both strains. Compared to wild-type C57BL/6 mice, analysis of leukocyte and lymphocyte subpopulations in the humanized mice indicates that the CD4/CD8 ratio is altered in favor of CD4+ T cells in both strains, similar to what was observed in B-HLA-A2.1 humanized mice. Despite these observed alterations, B-HLA-A24.2 mice were able to mount an immune response to peptide SART2 93-101, which was measured by splenocyte IFN-γ production. In summary, our data indicate that humanized HLA-A mice provide a powerful preclinical model for in vivo/ex vivo evaluation of novel peptide vaccines. Citation Format: Zhenlan Niu, Xuan Yu, Zhiyuan Shen, Jing Guo, Qingcong Lin, Hui Xu. Development and validation of humanized HLA mouse model platforms for preclinical evaluation of novel peptide vaccines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5345.

Abstract 4184: Change of oncological features on tumor and immune cells after anti-PD-1 antibody to pancreatic cancer orthotopic model in CD34+ HSC based humanized mice

Abstract (a) To investigate novel therapeutic agents in pancreatic cancer, animal models that recapitulate the features of the human immune system are necessary. We aim to analyze an orthotopic model in CD34+HSC-based humanized mice and to prove that is an indispensable model for immune-oncology therapy research. (b) We established an orthotopic tumor model using luciferase-expressing BxPC3 cells in humanized NSG mice. After inoculation of tumor cells, anti-PD-1 Ab was administered to mice 5 times a week by I.P. injection. Tumor formation and progression were monitored using IVIS. To compare the subsets and immune-check point receptors of human immune cells in blood and tumor tissue, Flow cytometry and immunohistopathology were used. Additionally, we compared the distribution of various cells included in the tumor and the mRNA expression level of each cell through a single cell RNA sequence. We also establish primary cell lines and organoids with the humanized mouse tumor for further evaluation. (c) After inoculation of BxPC3_Luc cell line into mice pancreas, we monitored tumor growth using a noninvasive imaging method of IVIS. When we compared the tumor growth between the anti-PD-1 treatment and control, there was no significant difference. Using FACS, we analyzed the various markers of human immune cells in mouse blood and tumor tissue. In blood, PD-1 expression of CD8+ T cells decreased, and Tim-3 expression increased in anti-PD-1 group. And, PD-1 expression of CD4+ T cells decreased in anti-PD-1 group. In tumor tissue, we also verified the decrease of PD-1 expression on tissue infiltrating CD8+ T cells and CD4+ T cells in the anti-PD-1 group. Especially in the more responsive mouse among anti-PD-1 group, activation markers in cytotoxic T cells (CD8+), helper T cells (CD4+), and regulatory T cells (CD4+FOXP3+CD25+) increased, and also, M1 macrophage (CD86+) expression was increased. Furthermore, we compared the mRNA expression levels of various cells in the tumor microenvironment according to anti-PD-1 Ab treatment through single cell RNA seq analysis. It was confirmed that the expression of genes related to proliferation and growing decreased, while the expression of genes related to apoptosis increased in cancer cells. Finally, we constructed primary cancer cells and organoids from tumor tissue and confirmed that the immune-oncological characters of these models are maintained after passaging. (d) We successfully established a pancreatic orthotopic model in a humanized mouse and analyzed the changes in immune cells and tumor cells after anti-PD-1 treatment. In addition, by obtaining tumor tissue-based preclinical models, we laid the foundation for future immune-oncologic experiments. We hope that these attempts and results will help in the development and drug verification of new immunotherapy agents for refractory solid tumors such as pancreatic cancer. Citation Format: Sujin Park, Eun-young Koh, Inha Yoo, Ji Hye Jeong, Eunsung Jun. Change of oncological features on tumor and immune cells after anti-PD-1 antibody to pancreatic cancer orthotopic model in CD34+ HSC based humanized mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4184.

Abstract 4186: Development of an orthotopic A549 lung carcinoma model in CD34+ humanized NCG mice and response to treatment with paclitaxel and pembrolizumab

Abstract Translational in vivo models are needed to expedite the discovery of new treatments for lung cancer. One such approach is the use of humanized mouse models that enable immunotherapies targeting human immune cells to be evaluated for efficacy and tolerability. We developed an orthotopic lung carcinoma model in humanized mice (HuCD34 NCG), utilizing serial bioluminescence imaging to evaluate the activity of paclitaxel and pembrolizumab as single and combination agents. Paclitaxel, which disrupts microtuble function, and pembrolizumab, a PD-1-targeting immunotherapy, are both approved for the treatment of certain types of lung cancer. Paclitaxel kills cancer cells directly, and may also promote anti-tumor responses through enhanced presentation of tumor antigens and activation of T cells. Immunodeficient NCG mice were humanized with cord blood-derived hCD34+ stem cells from 3 donors. High levels of human cell engraftment, with expected frequencies of hCD45+ cells and other immune subsets, was observed by 14 weeks post-injection. Luciferase expressing A549 cells were implanted orthotopically into the pulmonary space of the left lobe of the lung, and animals were subsequently flux sorted into treatment groups. Tumor burden, measured by luciferase expression, increased progressively in untreated animals, and moribundity was often associated with clinical indications of respiratory distress. Treatment with paclitaxel significantly decreased the tumor burden and extended survival. In contrast, pembrolizumab did not control tumor growth or extend survival. There was no synergistic effect with the combination therapy, in fact, the addition of pembrolizumab was associated with a modest increase in tumor burden and disease progression. Comparative analysis of immune cell composition, function and persistence in lung and other tissues provided insights into the distinct pharmacodynamic responses associated with the different treatment regimens. This model establishes a robust in vivo platform to identify novel therapies for the treatment of lung cancer. Citation Format: Anya Avrutskaya, Elizabeth Rainbolt, Bincy John, Cara Clouse, Murray Stackhouse, Christopher Currie, Sydney Scatigno, Steven Bronson, Jenny Rowe, Chassidy Hall, David P. Harris. Development of an orthotopic A549 lung carcinoma model in CD34+ humanized NCG mice and response to treatment with paclitaxel and pembrolizumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4186.

Abstract 6181: Digital pathology workflow: Building a bridge for translational biomarker development in humanized mice

Abstract The introduction of immune checkpoint inhibitors (ICIs) directed against programmed death protein 1 (PD-1), as well as cytotoxic T lymphocyte antigen 4 (CTLA-4), has revolutionized the treatment of non-small cell lung cancer (NSCLC) in the last decade. These therapies have shown significant success in a subset of NSCLC patients, leading to durable response and improved overall survival. However, clear predictive biomarkers for response are still yet to be defined. Tumor-infiltrating lymphocytes (TILs) as one important part of the tumor microenvironment (TME) have proven to be predictive biomarkers in other tumor types such as melanoma, whereas not fully understood in NSCLC. The assessment of TILs typically involves techniques such as flow cytometry (FC) or immunohistochemistry (IHC). While the former has the advantage of being quantitative and relatively easy to standardize it lacks translational value as most clinical samples will be examined using IHC. The current study aims to compare both methods at terminal end point and for treatment outcome, using a panel of 14 NSCLC PDX models, implanted in humanized NSG mice and treated with anti-CTLA4, anti-PD1 and the combination thereof. Based on FC analyses of the untreated tumor tissue the PDX models were stratified into seven hot (>5% TILs) and seven cold (<5% TILs) tumors. The IHC samples were analyzed by digital pathology for TIL infiltration by making use of automated segmentation of the three main tissue classes: tumor, stroma and necrosis. Small number of IHC slides were manually annotated to form a training set, which were then used to train a deep learning model using Visiopharm, a commercial AI platform for image analysis. By quantification of TILs in a spatial context of the tumor tissue, across all models, tumor was the most prominent tissue class. The tissue class distribution as well as the TIL rate (FC or IHC measure) is a model immanent feature. Under treatment, the ratio was stable in case of the cold tumors. In the hot tumors the stroma compartment increased while tumor tissue decreased. Treatment sensitive models displayed an increase of TILs confirmed by FC and IHC. The latter revealed that the TILs specifically increased in the tumor tissue whereas decreased or was stable in the stroma. This observation was significantly more pronounced in cold tumors compared to hot tumors (Oneway ANOVA, p < 0.001). Furthermore, a positive correlation between FC and IHC results on CD45 positive cells in the tumors (p< 0.002) could be confirmed. In this study, we proofed the feasibility of software-based image analysis as translational relevant read-out in preclinical animal experiments. The possibility to reproducibly quantify tissue classes, immune cell markers and including spatial information paves the way towards discovery of novel features predicting response in translational immuno-oncology research. Citation Format: Matti Hakkarainen, Timmo Bragge, Jussi Rytkönen, Eva Oswald, Julia B. Schueler. Digital pathology workflow: Building a bridge for translational biomarker development in humanized mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6181.

Abstract 4695: Effect of fully human anti-PTGFRN antibody drug conjugate on head neck cancer cells and patient derived xenografts

Abstract Prostaglandin F2 Receptor Negative regulator, or PTGFRN, also known as FPRP is a 175 kDa transmembrane protein associated with metastatic characteristics of certain cancer types and a member of the tetraspanin family. PTGFRN expression is undetectable to low in normal tissues whereas it is elevated in several cancers such as head and neck, mesothelioma and medulloblastoma. PTGFRN has also been reported to be upregulated in many different types of cancer, increasingly so in metastatic cancer cells, compared to their non-metastatic counterparts PTGFRN expression has been reported to be a poor prognostic factor for glioblastoma making it a potential novel cancer therapeutic target. We have previously shown that PTGFRN is an internalizable cell surface protein making it suitable for targeting by antibody drug conjugate (ADC). We have developed fully human monoclonal antibodies to PTGFRN by immunizing humanized transgenic TC mice with human PTGFRN. Within 60 days from the start of immunization we were able to select several fully human anti-PTGFRN monoclonal antibodies that were examined by several functional assays that included flow binding, internalization, affinity determination and cell killing as antibody drug conjugates. Several monoclonal antibodies were selected and evaluated for their ability to inhibit tumor formation in several cancers. Selected antibodies were then conjugated to duocarmycin by a cleavable linker. The present study proposed to examine the effect of one such anti-PTGFRN ADC on head and neck cancer cell lines and patient derived xenografts that had been analyzed for their PTGFRN expression by western blot analysis using anti-PTGFRN antibody. Cells lines and PDX that were positive for PTGFRN expression were used to examine the effect of anti-PTGFRN ADC on their in vitro and in vivo proliferation. The data show that anti-PTGFRN ADC inhibits tumor formation in a dose-dependent fashion. These data also establish PTGFRN as cancer druggable target and the selected fully human anti-PTGFRN antibody as an attractive antibody for the development of new antibody drug conjugate in Oncology. These data also emphasize the potential of humanized transgenic TC mice for the development of fully human monoclonal antibodies, thus bypassing the need for humanization and affinity maturation required by other humanized mice or phase display. Citation Format: Jorge Marquez, Jianping Dong, Mitsuo Oshimura, Jun Hayashi, Ginette Serrero. Effect of fully human anti-PTGFRN antibody drug conjugate on head neck cancer cells and patient derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4695.

Abstract 5346: A novel humanized CD16A model to evaluate efficacy of human antibody therapeutics in triggering ADCC

Abstract Antibody-dependent cellular cytotoxicity (ADCC) is a mechanism by which immune effector cells engage the Fc portion of antibodies bound to antigen on the surface of another cell, thereby targeting that cell for destruction. In mice, the Fcgr4 receptor is primarily responsible for triggering ADCC, while the FCGR3A receptor (CD16A) mediates this function in human NK cells, which are an attractive target cell type for cancer immunotherapy. Given these differences, we sought to generate humanized CD16A preclinical models to evaluate the efficacy of antibodies in triggering human CD16A-mediated ADCC in vivo. Previously, we established humanized CD16A mice on immunocompetent backgrounds for evaluating efficacy of novel antibodies in preventing growth of syngeneic tumor models. Here, we introduce a novel humanized CD16A mouse on the CB-17-SCID background, in order to inoculate human tumor cells for therapeutic efficacy testing. We confirmed expression of CD16A in human NK cells and monocytes/macrophages, but not granulocytes in the model; expression of murine Fcgr4 was no longer detectable, indicating successful generation of the model. Assessment of leukocyte populations in the humanized model indicated no significant alterations in the percentages of NK cells, dendritic cells, granulocytes, monocytes and macrophages in the spleen or blood of the humanized mice compared with wild-type CB-17 SCID mice. Subsequently, the ability of these mice to grow human tumor cells was evaluated: B-hHep3B-Luc, HC116, K-562, and NCI-N87 tumors were all successfully established. Finally, efficacy of an anti-human CLDN18.2 antibody was evaluated against B-hCLDN18.2 BxPC-3 tumors in the humanized mice, which showed modest anti-tumor efficacy at 30 mg/kg when compared to isotype control-treated mice. In summary, humanized B-hCD16A mice (CB-17-SCID) provide a suitable model for evaluating the ability of antibody candidates to induce ADCC. Citation Format: Xiao Liu, Zhenlan Niu, Ruili Lv, Qingcong Lin, Mari Kuraguchi, Qingqing Xu. A novel humanized CD16A model to evaluate efficacy of human antibody therapeutics in triggering ADCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5346.

Abstract 1420: Mechanism of NPRL2 gene therapy induced antitumor immunity in KRAS/STK11mt aPD1 resistant metastatic NSCLC

Abstract NPRL2/TUSC4 is a tumor suppressor gene whose expression is reduced in many cancers including NSCLC. Restoration of NPRL2 induces cell cycle arrest and apoptosis. We investigated the antitumor immune responses to NPRL2 gene therapy in aPD1 resistant KRAS/STK11mt NSCLC in a humanized mouse model. Humanized mice were generated by transplanting human cord blood-derived CD34 stem cells into NSG mice. Mice harboring > 25% human CD45 cells were considered humanized. Lung metastases were developed in humanized mice by injecting KRAS/STK11mt/aPD1R A549 cells, which were treated with i.v. injection of NPRL2 nanoparticles (DOTAP-NPRL2) with or without pembrolizumab (aPD1). NPRL2 treatment reduced lung metastases, whereas pembrolizumab was ineffective. The antitumor effect was greater in humanized than non-humanized mice suggesting the role of immune cells. The antitumor effect was associated with increased infiltration of human cytotoxic immune cells and decreased numbers of Treg in tumors. NPRL2+ pembrolizumab was not synergistic in this resistant model but was synergistic in KRASwt/aPD1S H1299 tumors. Cytotoxic immune cells in tumors were associated with the antitumor effect. Consistent with A549, NPRL2 showed a significantly strong antitumor effect on other KRASmt/aPD1R syngeneic LLC2 tumors whereas aPD1 was not effective. The antitumor effect of NPRL2 was correlated with HLA-DR+ DC, CD11c DC, TILs and NK cells in TME. The antitumor effect of NPRL2 was abolished upon in-vivo depletion of CD4, CD8 T, and MΦ in the LLC2 tumor model. However, no effect was found on in-vivo depletion of NK cells. Nanostring analysis on lung metastasis resulted in a distinct pattern of human gene expression by NPRL2. T cell functional genes, including IFNγ, CD8b, CD7, TNFSF18, ITGA1, GATA3 and TBX21 were significantly increased. Conversely, the negative regulatory genes, including FOXP3, TGFB1, TGFB2, and IL-10RA were inhibited. NPRL2 also downregulated T cell co-inhibitory molecules, including CTLA4, ICOS, LAG3, PDCD1, CD274, IDO1, PDCD1LG2, CD47, and KLRB1. Stably expressing NPRL2 clones were established, and tumors in humanized mice with these clones exhibited significantly slower growth compared to controls. TME analysis showed an upregulation of human CD45, CD3, CD8 T, HLA-DR+ DC and a downregulation of Tregs, CD3+PD1+T, MDSC, and CD163+ TAM in tumors expressing NPRL2. The stable cells showed a substantial increase in both colony formation inhibition and apoptosis. Stable clones showed heightened sensitivity to carboplatin in colony formation, apoptosis, and PARP cleavage assays. Stable expression of NPRL2 resulted in the downregulation of both AKT-mTOR and MAPK pathways by inhibition of pAKT, pmTOR, pPRAS40, p4E-BP1, pS6, and pERK1/2. Taken together, NPRL2 gene therapy induces antitumor activity on KRAS/STK11mt/aPD1R tumors through DC-mediated antigen presentation and cytotoxic immune cell activation Citation Format: Ismail M. Meraz, Mourad Majidi, Renduo Song, Feng Meng, Lihui Gao, Qi Wang, Jing Wang, Elizabeth Shpall, Jack A. Roth. Mechanism of NPRL2 gene therapy induced antitumor immunity in KRAS/STK11mt aPD1 resistant metastatic NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1420.

Abstract 5464: Galectin-3 expression in Ewing sarcoma: Modulation of the immune microenvironment in an immunocompetent model

Abstract Objective: Ewing sarcoma is an aggressive cancer of adolescents. Patients with metastatic Ewing sarcoma often receive radiation as part of their treatment protocol. We have demonstrated that both Ewing tumor cells and T cells infiltrating Ewing tumors express Galectin-3 (Gal3), an immunosuppressive protein in the tumor microenvironment. Here, we utilize an immunocompetent, humanized mouse model of Ewing sarcoma to investigate the role of Galectin-3 in the Ewing tumor immune microenvironment both at baseline and following radiation. Specifically, we sought to determine the influence of Galectin-3 on post-radiation inflammation in Ewing sarcoma. Methods: Human Ewing cells -/+ Gal3 expression were generated using CRISPR/Cas 9 technology. Cells -/+ Gal3 were orthotopically injected into humanized mice and allowed to established tumors over ~3 weeks. Tumors were then treated with radiation (or no radiation controls). To also address immune cell expression of Gal3, galectin inhibitors were utilized. Tumors were analyzed by bulk RNA sequencing and flow cytometry analysis (Cytek Aurora) for human CD45, CD3, CD4, CD20, CD8, CD56, CD14, CD16, etc. Peripheral blood controls were also collected and analyzed to ensure consistent baseline immune reconstitution. Results: CHLA10 and TC32 Ewing sarcoma cell lines both demonstrate Gal3 expression at baseline by western blot. CRISPR/Cas 9 knock-out of Gal3 in both cell lines showed successful knock down. Impact on growth and sensitivity to radiation in vitro was assessed by live cell Incucyte assays. Tumors in humanized mice were established utilizing these cells (or Gal3 expressing non-targeting controls) and changes in the post-radiation transcriptional profiles were assessed. Immune cells infiltrating these tumors were also quantified. Ongoing work focuses on additional characterization of these tumor infiltrating immune cell populations following radiation therapy and the impact of broader galectin inhibition on this effect. Conclusion: This work contributes to the understanding of radiation-induced tumor inflammatory responses in Ewing sarcoma by defining the role of tumor and T cell galectin-3 expression inn Ewing immunobiology. Next steps will explore translational opportunities based on these findings. Citation Format: Yunash Maharjan, Sreya Dey, Elina Mukherjee, Jessica Daley, Kelly M. Bailey. Galectin-3 expression in Ewing sarcoma: Modulation of the immune microenvironment in an immunocompetent model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5464.

Abstract 3255: Targeting CD4+FOXP3+ Tregs to enhance anti-tumor immunity

Abstract We strive to improve the outcomes of cancer immunotherapy by using a new generation of chemically modified self-delivering antisense oligonucleotides (ASO), termed FANA ASO, to target the key T-regulatory (Treg) cell transcription factor, FOXP3. We tested five murine FANA in 2 murine tumor models (TC1, MC38) and studied 19 human FANA FOXP3 in vitro with human healthy donor PBMC, followed by use of clinical samples from lung cancer patients and patients with mesothelioma, as well as testing in humanized mice (hu-PBMC-NSG). In murine studies, we treated mice on day 7 after tumor inoculation with Scramble control or murine FANA FOXP3, 50 mg/kg for 14 days, i.p. In the TC1 lung tumor model, we found an average 38% decrease of tumor volumes, p = 0.0007, and 22% of tumors were completely resorbed. In the MC38 colon carcinoma model, we found a 49% decrease of tumor volumes, p = 0.0182, and 13.6% of tumors were completely resorbed. In both tumor models, we observed an ~50% decrease of Foxp3 mRNA by qPCR, and decreased numbers of intratumoral Tregs by flow cytometry. This was accompanied by significant decreases (p<0.05) of the mRNA and protein intratumoral levels of multiple exhaustion markers: LAG-3, CTLA-4, PD-1, Tim-3, Tigit, CD244, CD160 and VISTA, and increased expression of perforin and granzyme-b by intratumoral T cells. In addition, there were no changes in FOXP3 mRNA expression or in the numbers of Tregs in draining lymph nodes and in spleens of tumor bearing mice, suggesting that intratumoral Treg have enhanced sensitivity to FANA FOXP3 in vivo. We identified the best 5 of 19 human FANA FOXP3 and showed these had no toxic effects on cell viability or division, and downregulated FOXP3 expression over 50% in PBMC from healthy donors (p<0.05). Human Treg, pre-incubated with FANA FOXP3 for 3 hours, retained only 60.3% of their suppressive function in vitro. FANA FOXP3 treatment of lung cancer tumor samples and mesothelioma pleural effusion samples resulted in a 56.4% decrease in FOXP3 mRNA expression and a 61.5% decrease in Treg numbers (all p<0.05). Moreover, tumor FOXP3+ Treg had 36.7% less FOXP3 protein per cell, and downregulated CD39 expression compared to Scramble control. Targeting of cancer samples with human FANA FOXP3 in vitro was associated with increased division of CD4+ and CD8+ cells, and with downregulation of multiple exhaustion molecules, including CTLA-4, Tim-3, PD-1, LAG-3 and TIGIT. Humanized mice, treated with two human FANA FOXP3 compounds (50 mg/kg, 7days), demonstrated an absence of apparent toxic effects and human FOXP3 mRNA was downregulated by 47.4% in the blood, by 74.2% in lymph nodes and by 51.8% in the spleens (all p<0.05). Hence, targeting of intratumoral Tregs using FANA FOXP3 results in enhanced immune responses to solid tumors without inducing autoimmunity, and studies are underway in combination with checkpoint inhibitors and/or CAR-T cell therapy. Citation Format: Tatiana Akimova, Liqing Wang, Zhanna Bartosh, Evgeniy Eruslanov, Steven Albelda, Sunil Singhal, Veenu Aishwarya, Wayne W. Hancock. Targeting CD4+FOXP3+ Tregs to enhance anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3255.

Abstract 3960: Establishment of humanized mouse models engrafted with luciferase-expressing acute myeloid leukemia cell lines

Abstract Introduction: Because acute myeloid leukemia (AML) is a very aggressive and quickly developing cancer, for which actual treatments are either inefficient, poorly tolerated, or leading to resistance, there is a strong need for an effective platform to evaluate novel therapies. Our aim is to provide a relevant humanized mouse model as a precious tool to evaluate innovated treatments to cure AML. Methods: Mice were humanized with CD34+ hematopoietic stem cells to reconstitute a complete human immune system. Three AML cell lines, namely OCI-AML2, OCI-AML3 and MOLM-14, all expressing mCherry and luciferase, were intravenously engrafted at different doses in non-humanized and CD34-humanized mice. Tumor proliferation was monitored by bioluminescence imaging (BLI) thanks to luciferase expression on tumor cells. Their expression of mCherry was also exploited to determine the presence of tumor cells in blood and tissues using flow cytometry. Results: All AML cell lines engrafted in mice, but we observed different growth kinetics and homogeneity depending on the humanization of mice and the dose injected. Indeed, tumor grew more slowly and heterogeneously in humanized mice, which underlines the role of human immune cells reconstituted in mice. This was improved by increasing the number of tumor cells injected. Tumor cells were found in femurs, spleen, kidneys, ovary, lungs, and uterus depending on the cell line. Mice survival was also highly affected by the tumor cell line engrafted. We observed that MOLM14 was the most aggressive model, while OCI-AML3 reached a steady state and were tolerated by mice over a long period. Conclusion: We established 3 different AML cell lines in a model of humanized mice which nicely recapitulates the growth of tumor cells in target organs. These 3 models behave differently in terms of growth kinetic, tolerability by mice, and organs infiltrated, which gives plenty of opportunities to evaluate novel therapies designed to cure AML. Citation Format: Emilie Bayon, Arije Mersni, Audrey Wetzel, Jade Villé, Kiave Yune Ho Wang Yin. Establishment of humanized mouse models engrafted with luciferase-expressing acute myeloid leukemia cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3960.

Abstract 4191: Delayed onset of GvHD in PBMC humanized NCG-B2m KO mice provides an enhanced model for oncology studies

Abstract Humanized mice are specialized animal models incorporating components of the human immune system. These models are valuable tools for assessing the efficacy of human immunotherapies on tumors. Humanized mice are created by injecting human CD34+ hematopoietic stem cells (HSCs) or human peripheral blood mononuclear cells (PBMCs) into an immunodeficient mouse. PBMC humanized (HuPBMC) mice engraft within a few weeks and can recapitulate a robust human T cell population. One challenge when using HuPBMC mice is the onset of graft versus host disease (GvHD), limiting their lifespan compared to hCD34+ HSC humanized mice. The onset of GvHD varies in the HuPBMC model depending on the donor cells. The development of xenogeneic GvHD is dependent on the expression of major histocompatibility complex (MHC) on host cells and lowering MHC expression can delay the onset of GvHD.NCG-B2m KO mice lack beta-2 microglobulin, a component of MHC I, and have lower cell surface expression of MHC I molecules. This leads to a decrease in recognition of host cells by human immune cells. Here we compared the onset of GvHD between HuPBMC NCG-B2m KO mice and HuPBMC NCG mice. PBMC donor variability was also compared between the two PBMC humanized models by evaluating multiple donors and assessing additional collections from the same donor. After the PBMC engraftment was characterized, HuPBMC NCG mice and HuPBMC NCG-B2m KO mice were enrolled in tumor studies comparing outcomes and tumor growth kinetics in mice treated with human immune checkpoint blockade therapy.NCG and NCG-B2m KO mice were injected with 1x107 PBMCs from individual donors. Animals were weighed three times a week to monitor for changes in body weight and general health status. Peripheral blood was collected at day 10, 20, 30, 40, 60 and 90 post injection for flow cytometry screening of human immune cell engraftment (hCD45+, hCD3+, hCD4+ and hCD8+). Human PBMCs isolated from additional collections from the same donors were also injected into both NCG-B2m KO mice and NCG mice and flow cytometry was performed. HuPBMC NCG-B2m KO mice survived longer than HuPBMC NCG mice allowing for the potential use in longer term tumor studies before the results are confounded by GvHD. PBMC humanized mice are an important model for translational research using immunotherapies and the PBMC humanized NCG-B2m KO model provides an expanded study window ideal for oncology studies. Citation Format: Steven Bronson, David Harris, Anya Avrutskaya, Christoph Eberle, Jenny Rowe. Delayed onset of GvHD in PBMC humanized NCG-B2m KO mice provides an enhanced model for oncology studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4191.

Abstract 7503: Non-clinical study of circulating tumor cells in humanized mice: Post-xenograft effects in immunodeficient mice

Abstract Circulating tumor cells (CTCs) possess the potential to serve as innovative markers in clinical trials, contributing to the understanding of cancer metastasis mechanisms, diagnosis, prognosis, the development of personalized treatment strategies, and enabling the monitoring of treatment efficacy. NSG mice, which are a strain of immunodeficient mice frequently utilized as a non-clinical model for cancer research, exhibit diminished levels of natural killer (NK) cells and abnormal monocytes in their bloodstream. In a previous study, a positive correlation was observed between the duration of injection and the levels of white blood cells (WBC) in mice that were injected with various cancer cell lines derived from humans. To evaluate the correlation among the primary tumor and circulating tumor cells (CTCs), as well as the impact on immune cells, we assessed changes in immune cell composition, primary tumor size, and enumeration of CTCs in xenografted mice over a period of time. This investigation was carried out using CytoGen's Smart Biopsy™ systems. The results, which were obtained using equipment including CytoGen's Smart Biopsy™ Systems, showed that the distribution of white blood cell (WBC) components exhibited variations based on age, primary tumor burden, and circulating tumor cell (CTC) count following injection. A notable association was observed between the augmentation of immune cells and the quantity of circulating tumor cells (CTCs) present in the bloodstream. Therefore, alterations in immune cells may potentially impact the potential to affect the ability to influence the efficacy of cancer immunotherapy, metabolic therapy, and anti-metastatic therapy. When conducting non-clinical investigations with the objective of developing cancer treatment protocols, it is crucial to meticulously select humanized or immunodeficient mice that are appropriate for the study. Therefore, it is advisable to perform regular monitoring of murine blood samples in order to observe any variations in the counts of circulating tumor cells (CTCs), while considering a standardized blood volume per individual. In the immunodeficient mouse model, the existence of circulating tumor cells (CTCs) results in an increase in the quantity of cells present in the bloodstream. The observed rise in circulating tumor cells (CTCs) is found to be concurrent with the progression and development of the primary tumor. The consideration of this aspect is of utmost importance in the formulation of non-clinical studies aimed at the development of cancer treatment protocols. Citation Format: Sehyung Pak, Hajin Pak, Hee Jin Yoo, Sra Min, Hui Ji Kim, JaeYoon Lee, Min-Ji Song, Jaesung Choi, Aejin Jeong, Hanbyeol Kim, Jung Won Kim, Hai-Chon Lee. Non-clinical study of circulating tumor cells in humanized mice: Post-xenograft effects in immunodeficient mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7503.

Abstract 6429: Critical role of elevated TMEM176B gene expression in humanized patient-derived xenografts mouse models of triple negative breast cancer

Abstract Objective: In the cancer research, a highly promising in vivo platform for personalized treatment involves the adoption of an advanced approach—specifically, the grafting of patient-derived xenografts (PDX) onto humanized mouse models. These models capture distinctive aspects of interaction with the human immune system and the tumor microenvironment (TME). In this study, we have engineered humanized triple-negative breast cancer (TNBC) PDX models to observe alterations in differentially expressed genes, thereby enhancing our understanding of the intricacies of personalized treatment in this context. Method: We utilized two TNBC PDX models established for the humanized models, where CD34+ hematopoietic stem cells were transplanted into immunodeficient mice. RNA sequencing was performed to identify altered gene expression in humanized PDX models compared to NSG PDXs. Additionally, we generated stable cell lines overexpressing TMEM176B in MDAMB231 and Hs578T cells, and we knocked down TMEM176B in 4T1 cells using recombinant techniques Results: In the humanized model, the growth of PDX tumors did not show a significantly faster or markedly different rate compared to previous studies. However, upon conducting RNA sequencing in the PDX model, the expression of the TMEM176B gene was consistently downregulated. Consequently, we transfected and observed that the increased expression of TMEM176B in MDAMB231 and Hs578T cell lines resulted in enhanced cell proliferation in vitro. Transplanting MDAMB231 cells overexpressing TMEM176B into athymic nude mice resulted in accelerated tumor growth. The study further investigates the expression of TMEM176B in an immune-competent environment by knocking down the gene in 4T1 cells or transplanting the control group into a balb/c mouse model. Tumors with TMEM176B knockdown exhibited inhibited growth, increased CD3+ T cell infiltration, and significant inhibition when treated with anti-PD-L1 compared to the control group. Conclusion: This research demonstrates the utility of PDX models, particularly humanized PDX models, in studying the complexities of cancer biology and the tumor microenvironment. The findings regarding TMEM176B and its role in tumorigenesis and immune responses in triple-negative breast cancer provide valuable insights for further research and potential therapeutic interventions. Citation Format: Yujeong Her, Jihui Yun, Woohang Heo, Jong-Il Kim, Han-Byoel Lee, Wonshik Han, Hyeong-Gon Moon. Critical role of elevated TMEM176B gene expression in humanized patient-derived xenografts mouse models of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6429.