Human Xenograft Mouse Model Research Articles

Tumor-targeted delivery of SNHG15 siRNA using a ZIF-8 nanoplatform: Towards a more effective prostate cancer therapy

Small interfering RNAs (siRNAs) can be used as a powerful tool in gene therapy to downregulate the expression of specific disease related genes. Some properties however, such as instability, and low penetration into cells can limit their efficacy, and thus reduce their therapeutic potential. Metal-organic frameworks (MOFs) such as zeolitic imidazolate framework-8 (ZIF-8), which consist of organic bridging ligands and metal cations (Zn), have a very high binding affinity with nucleic acids including siRNAs. In this study, we designed a PEGylated ZIF-8 platform that was equipped with epithelial cell adhesion molecule (EpCAM) aptamer for the targeted delivery of siRNA molecules, in order to knockdown SNHG15 in both a prostate cancer (PC) cell line, and a human PC xenograft mouse model. SNHG15 is a long noncoding RNA, with oncogenic roles in different cancers including PC. The results indicated that the depletion of SNHG15 by Apt-PEG-siRNA@ZIF-8 nanoplatfrom inhibited cell proliferation and colony formation, and increased apoptosis in PC cells. This nanoparticle facilitated the release of siRNAs into the tumor environment in vivo, and subsequently reduced the tumor growth, with no side effects observed in vital organs. We have therefore developed a novel siRNA nano-delivery system for targeted prostate cancer treatment; however further studies are required before it can be tested in clinical trials.

Development of therapeutic monoclonal antibodies against DKK1 peptide-HLA-A2 complex to treat human cancers

BackgroundTargeted immunotherapy with monoclonal antibodies (mAbs) is an effective and safe method for the treatment of malignancies. Development of mAbs with improved cytotoxicity, targeting new and known tumor-associated antigens, therefore...

CD40 is expressed in the subsets of endothelial cells undergoing partial endothelial-mesenchymal transition in tumor microenvironment.

Tumor progression and metastasis are regulated by endothelial cells undergoing endothelial-mesenchymal transition (EndoMT), a cellular differentiation process in which endothelial cells lose their properties and differentiate into mesenchymal cells. The cells undergoing EndoMT differentiate through a spectrum of intermediate phases, suggesting that some cells remain in a partial EndoMT state and exhibit an endothelial/mesenchymal phenotype. However, detailed analysis of partial EndoMT has been hampered by the lack of specific markers. Transforming growth factor-β (TGF-β) plays a central role in the induction of EndoMT. Here, we showed that inhibition of TGF-β signaling suppressed EndoMT in a human oral cancer cell xenograft mouse model. By using genetic labeling of endothelial cell lineage, we also established a novel EndoMT reporter cell system, the EndoMT reporter endothelial cells (EMRECs), which allow visualization of sequential changes during TGF-β-induced EndoMT. Using EMRECs, we characterized the gene profiles of multiple EndoMT stages and identified CD40 as a novel partial EndoMT-specific marker. CD40 expression was upregulated in the cells undergoing partial EndoMT, but decreased in the full EndoMT cells. Furthermore, single-cell RNA sequencing analysis of human tumors revealed that CD40 expression was enriched in the population of cells expressing both endothelial and mesenchymal cell markers. Moreover, decreased expression of CD40 in EMRECs enhanced TGF-β-induced EndoMT, suggesting that CD40 expressed during partial EndoMT inhibits transition to full EndoMT. The present findings provide a better understanding of the mechanisms underlying TGF-β-induced EndoMT and will facilitate the development of novel therapeutic strategies targeting EndoMT-driven cancer progression and metastasis.

TREM2 Agonism Affects Human Microglia Response in the Presence of Amyloid Pathology In Vivo

AbstractBackgroundPharmacological activation of TREM2 represents a novel therapeutic approach to slow Alzheimer´s disease (AD) progression. Mutations in microglial gene, TREM2, confers increased risk to developing late‐onset AD. Moreover, animal studies demonstrate the loss‐of‐of function variants or complete absence of Trem2 aggravates amyloid pathology. Genetic ablation of TREM2 function has been shown to lock microglia in a homeostatic state, preventing a switch to the disease‐associated state supporting the phagocytic clearance of misfolded proteins and cellular debris. Due to low gene sequence and protein structure homology between mouse and human TREM2, as well as phenotypical disparities between mouse and human microglia, monitoring the impact of TREM2 activation in vivo requires humanized animal models.MethodHere we studied the impact of TREM2 agonism on xenografted human microglia in a mouse model of amyloid pathology. We systematically treated 7‐month‐old transgenic AppNL‐G‐F mice transplanted with human embryonic‐stem‐cell‐derived microglia with TREM2 small molecule agonists. Human microglia were sorted from the brain and subsequently assessed using multi‐omics technologies.ResultThe activation of TREM2 lead to increased expression of cytokines and chemokines in xenografted human microglia in the presence of amyloid pathology in AppNL‐G‐F, but not in wild‐type mice. Other changes in the transcriptome profile indicated shifts in microglia populations in TREM2‐agonist‐treated AppNL‐G‐F mice. These effects were observed at exposures consistent with activation of TREM2 in vitro assessed by pSYK and sTREM2 levels.ConclusionAll together, we show the human microglia xenograft mouse model is a valuable tool to study the impact of pharmacological TREM2 activation in the presence of AD neuropathology.

API-192, an Allogeneic CD19/20 CAR-NKT Cell Product Derived from Cord Blood CD34+ HSPCs for the Treatment of B-Cell Malignancies

Autologous chimeric antigen receptor (CAR)-T cell therapies induce durable remissions and improve survival in patients with advanced B cell malignancies. However, challenges such as manufacturing variability, cost, and time to treatment highlight the need for an allogeneic, off-the-shelf approach that preserves the efficacy of autologous CAR-T. Additionally, antigen escape is one of the established mechanisms of resistance to current CAR-T therapies which target a single antigen. Classical natural killer T (NKT) cells express an invariant T cell receptor (iTCR) that is restricted to the conserved CD1d protein, making it a promising cell type for allogeneic therapies as they do not require TCR gene editing to avoid graft-vs-host (GvH) disease. Furthermore, NKT cells mediate dendritic cell cross-priming, target immunosuppressive myeloid cells/macrophages in the tumor microenvironment, and directly kill CD1d + tumor cells through the iTCR. Endogenous NKT cells are an extremely rare population in the blood (<1% of lymphocytes), posing manufacturing challenges. API-192 is a human cord blood CD34 + hematopoietic stem and progenitor cell-derived NKT cell product engineered to express dual CARs targeting CD19 and CD20 and armored with soluble IL-15 for improved expansion and persistence. The Appia Cells Utilized for Allogeneic (ACUA) platform enables the production of large numbers of product NKT cells co-expressing multiple transgenes at high purity (>90% iTCR + 19CAR + 20CAR +) with no purification step or gene editing. Phenotypically, API-192 cells are predominantly CD4 -CD8 - or CD4 -CD8 +, express major activating NK receptors, and harbor intracellular stores of cytotoxic molecules, resembling endogenous NKT cells. API-192 kills Raji and Nalm6 tumor cells in a CAR-specific and dose-dependent manner and can control multiple rounds of tumor challenge in vitro. API-192 secretes pro-inflammatory cytokines and proliferates in response to tumor antigen . In human tumor xenograft models in immunocompromised mice, API-192 robustly expands, suppresses tumor growth, and exhibits prolonged persistence, enabling control of a tumor rechallenge 70 days after initial dosing. API-192 expresses lower levels of MHC I and MHC II molecules relative to conventional CAR-T cells and elicits less IFN-γ release by NK and T cells in mixed lymphocyte reaction (MLR) assays, indicative of a more favorable immunogenic profile. Compared to conventional CAR-T cells, API-192 also secretes minimal levels of IFN-γ in response to co-culture with healthy donor peripheral blood mononuclear cells, consistent with the expression of a non-HLA restricted TCR and low GvH risk. These results demonstrate the feasibility, potency, and potential safety of API-192, warranting further development as a novel allogeneic therapy.

Dual Targeting Novel WDR5/ATAD2 Oncogenic Signaling through CK2/Ikaros Axis Demonstrates Synergistic Efficacy in T-ALL

Background WD repeat-containing protein 5 (WDR5) is highly expressed in human cancers and its high expression is linked with adverse prognoses. However, the oncogenic roles of WDR5 and underlying mechanisms are undefined in T-cell acute lymphoblastic leukemia (T-ALL). Casein Kinase II (CK2) is a pro-oncogene, and CK2-mediated IKAROS dysfunction plays critical roles in high-risk ALL, and CK2 inhibitor CX-4945 has the anti-leukemic effect by restoring IKAROS function. This study aims to explore the synergistic anti-leukemic efficacy of dual targeting novel WDR5/ATAD2 oncogenic signaling via the CK2/IKAROS axis in T-ALL. Methods Clinical samples from 38 T-ALL patients and 32 healthy controls were obtained from Zhongda Hospital Southeast University. The expression of WDR5 and ATAD2 were tested by q-PCR and Western blot. RNA-seq was performed after CEM cells were treated with WDR5 inhibitor (OICR-9429), CX-4945 and vehicle control for 72 hours. The human leukemia xenograft mouse model is established by intravenously injected with WDR5 knockdown CEM (CEM-shWDR5) cells and shRNA control (CEM-shNC) into NSG mice. Mice were treated for 25 days in 4 groups [CEM-shNC + vehicle, CEM-sh WDR5 + vehicle; CEM-shNC + CX-4945 (100 mg/kg via gavage), CEM-sh WDR5 + CX-4945]. Leukemia burden in mice spleen and bone marrow was analyzed by flow cytometry. The survival of mice was compared by the Kaplan-Meier method. The synergistic effect of CX-4945 and OICR-9429 was analyzed by CCK8 assay. This study was approved by the Ethics Committee of Zhongda Hospital Southeast University Results WDR5 ishighly expressed in T-ALL patients compared with normal controls( P<0.001) and is higher in the relapsed samples than paired newly-diagnosed controls ( P<0.05) (Fig. 1A-B). WDR5 knockdown in T-ALL cells significantly suppressed cell proliferation and induced cell cycle arrest in vitro ( P<0.01) (Fig. 1C), and in vivo leukemia development (Fig. 1D). Also, WDR5 inhibitor OICR-9429 induces cell proliferation and cell cycle arrest in the cells. RNA-seq revealed that ATAD2 is downregulated upon OICR-9429 treatment and indicated that ATAD2 is the downstream target of WDR5. WDR5 knockdown suppresses ATAD2 expression ( P<0.01) (Fig. 1E). In addition, ATAD2 ishighly expressed in T-ALL patients versus normal controls ( P<0.05) (Fig. 1F), and ATAD2 silencing significantly suppresses cell proliferation and induces cell cycle arrest of T-ALL cells ( P<0.01) (Fig. 1G). These data indicate WDR5/ATAD2 signaling act as a novel potential oncogenic pathway in T-ALL. Furthermore, we found that IKAROS transcriptionally suppresses WDR5 expression, and CK2-mediated hyperphosphorylation of IKAROS is one of the major mechanisms of IKAROS dysfunction in ALL. CX-4945 strongly suppresses WDR5 expression in an IKAROS-dependent manner (Fig. 1H-J). The synergistic effect of CX-4945 with WDR5 disruption on cell proliferation suppression and cell cycle arrest was observed in T-ALL both in in vitro and in vivo xenograft mouse model. The combination of CX-4945 with WDR5 disruption significantly prolonged the survival of mice compared with single CX-4945 [55(52-58) days vs. 38.5(36-41) days， P<0.001] and single WDR5 disruption control [55(52-58) days vs. 41.5(39-44) days, P<0.001] (Fig. 1K). Consistently, the combination significantly reduces the size and weight of the spleen (Fig. 1L), % human CD45+ T-ALL cells in the spleen (Fig. 1M) and bone marrow (data not shown), and the expression of WDR5 and ATAD2 in the spleen versus single treatment controls ( P<0.01) (Fig. 1N-P). Finally, a synergistic effect of CX-4945 with OICR-9429 on cell proliferation suppression and down-regulation of WDR5 and ATAD2 was observed in the primary leukemic cells from T-ALL patients (Fig. 2A-D). Together, these data indicated the synergistic efficacy of CX-4945 with OICR-9429 on T-ALL and the novel model was summarized in Fig.2E. Conclusion In this study, we identified a novel WDR5/ATAD2 oncogenic signaling regulated by CK2/IKAROS axis in T-ALL. Our study reveals a model that dual targeting WDR5/ATAD2 signaling through direct inhibiting oncoproteins and via CK2/IKAROS axis to transcriptionally repress the oncoprotein to achieve synergistic efficacy. Our results further highlight the combination of CX-4945 with WDR5 inhibition is a potential option for the therapy of T-ALL patients.

Inhibition of transforming growth factor-β signals suppresses tumor formation by regulation of tumor microenvironment networks.

The tumor microenvironment (TME) consists of cancer cells surrounded by stromal components including tumor vessels. Transforming growth factor-β (TGF-β) promotes tumor progression by inducing epithelial-mesenchymal transition (EMT) in cancer cells and stimulating tumor angiogenesis in the tumor stroma. We previously developed an Fc chimeric TGF-β receptor containing both TGF-β type I (TβRI) and type II (TβRII) receptors (TβRI-TβRII-Fc), which trapped all TGF-β isoforms and suppressed tumor growth. However, the precise mechanisms underlying this action have not yet been elucidated. In the present study, we showed that the recombinant TβRI-TβRII-Fc protein effectively suppressed invitro EMT of oral cancer cells and invivo tumor growth in a human oral cancer cell xenograft mouse model. Tumor cell proliferation and angiogenesis were suppressed in tumors treated with TβRI-TβRII-Fc. Molecular profiling of human cancer cells and mouse stroma revealed that K-Ras signaling and angiogenesis were suppressed. Administration of TβRI-TβRII-Fc protein decreased the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), interleukin-1β (IL-1β) and epiregulin (EREG) in the TME of oral cancer tumor xenografts. HB-EGF increased proliferation of human oral cancer cells and mouse endothelial cells by activating ERK1/2 phosphorylation. HB-EGF also promoted oral cancer cell-derived tumor formation by enhancing cancer cell proliferation and tumor angiogenesis. In addition, increased expressions of IL-1β and EREG in oral cancer cells significantly enhanced tumor formation. These results suggest that TGF-β signaling in the TME controls cancer cell proliferation and angiogenesis by activating HB-EGF/IL-1β/EREG pathways and that TβRI-TβRII-Fc protein is a promising tool for targeting the TME networks.

Injectable Hydrogels Prepared Using Novel Synthetic Short Peptides with Defined Structure and Gelatin as Scaffolds to Support Cell and Tissue Growth.

Animal-derived basement-membrane matrices such as Geltrex are used to grow cells and tissues. Particularly, these are commonly applied to support tumor growth in animals for cancer research. However, a material derived from an animal source has an undefined composition, and may thus have unavoidable batch-to-batch variation in properties. To overcome these issues, a series of synthetic short peptides to form hydrogels is designed in combination with gelatin to promote cell adhesion and growth. The peptides have sequences of (X1Y1X2Y2)2 , where X1 and X2 are hydrophobic residues, while Y1 and Y2 are hydrophilic residues. The peptides spontaneously fold and self-assemble into a β-sheet secondary structure upon contact with salts, and then aggregate to form hydrophilic networks of hydrogels. Hybrid hydrogels formed by mixing the peptide IEVEIRVK (IVK8) with gelatin are injectable and enzymatically degradable. The hybrid hydrogels at optimal compositions support SW480 and HepG2 tumor spheroid growth in vitro as effectively as Geltrex. More importantly, the peptide/gelatin hydrogels support tumor growth in a SW480 human colorectal adenocarcinoma xenograft mouse model. Altogether, the results illustrate that the synthetic peptide/gelatin hybrid hydrogel is a promising scaffold that can be used to support cell and tissue growth both in vitro and in vivo.

Acquired Venetoclax Resistance in an In Vivo Model of B-Cell Precursor Acute Lymphoblastic Leukemia Is Characterized By Altered Functions of Apoptosis Regulators

Deregulation of cell death pathways such as apoptosis is a hallmark of cancer and contributes to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Apoptosis is controlled by different pro- and anti-apoptotic molecules either inducing or counteracting cell death signaling. Inhibition of anti-apoptotic molecules of BCL-2 family cell death inhibitors has been developed as therapeutic strategy. Venetoclax (VEN) binds selectively to BCL-2 resulting in apoptosis induction and has shown clinical activity in hematologic malignancies, but primary and secondary resistance have also been described. In this study, we aimed to identify resistance mechanisms acquired during VEN treatment leading to therapy failure of BCL-2 inhibition. In order to mimic a clinical situation with consecutive treatment cycles, we employed a patient-derived human BCP-ALL xenograft mouse model with subsequent courses of VEN administration to ALL bearing recipients in vivo. First, we estimated VEN-sensitivities in a series of BCP-ALL PDX samples (N=30) by ex vivo titration of individual VEN-EC 50 values and identified a range of heterogeneous VEN-activities. A VEN-sensitive (low VEN-EC50) PDX-ALL sample with a pro-B ALL phenotype carrying a KMT2A:: MLL1 gene fusion was selected and transplanted onto immunodeficient NOD/SCID recipient mice. Upon leukemia manifestation (presence of 5% human ALL cells in peripheral blood), mice were either treated with VEN (100 mg/kg, two times five days) or vehicle control. Recipients were sacrificed upon onset of leukemia-related morbidity and ALL cells isolated from spleens were transplanted onto secondary recipients, which were again treated with VEN or vehicle. ALL cells isolated from secondary recipients were retransferred onto tertiary mice and again exposed to in vivo VEN-treatment providing a total of three subsequent transplantation and in vivo VEN-therapy cycles. Assessing VEN sensitivities of the ALL cells initially transplanted and isolated after VEN-treatment cycles by using an ex vivo cytochrome c release assay showed increasing VEN-resistance. Accordingly, comparison of VEN to control in vivo treated cells showed significantly decreased sensitivities over treatment cycles. Importantly, also the time periods between treatment and leukemia re-occurrence in mice indicating decreasing VEN-sensitivity became shorter with every additional in vivo therapy course. Paired RNA- seq analysis revealed that transcript levels of the pro-apoptotic molecules BAX and BIM were significantly downregulated in VEN-exposed/resistant ALL as compared to vehicle control treated cells. Basal BH3 profiling analysis showed impaired apoptotic priming (significantly lower cytochrome c release after BIM and PUMA peptide exposure) and decreased dependence on anti-apoptotic BCL-2 and MCL-1 indicating a shift from a pro-apoptotic to a pro-survival state. This shift was also reflected in an e x vivo drug treatment assay showing decreased sensitivity to the MCL-1 inhibitor S63845 and the BCL-XL inhibitor A-1331852 in ALL cells from VEN- treated mice compared to control- treated mice (S63845 EC 50 1.5 vs. 2.2 µM, A-1331852 EC 50 9.3 vs. 15.3 µM). In line with the low BAX levels in ALL cells with acquired VEN-resistance, PDX BCP-ALL samples with intrinsic VEN resistance also showed lower BAX protein expression. Taken together, acquired VEN-resistance was recapitulated in a co-clinical trial model of BCP-ALL with repeated in vivo treatment cycles showing lower drug sensitivities along with increasingly reduced in vivo anti-ALL activity of VEN. Characterization of acquired VEN-resistance revealed decreased functional dependency on anti-apoptotic proteins and downregulation of pro-apoptotic BIM and BAX, thus pointing to an imbalance of pro- and anti-apoptotic molecules, which can be potentially targeted by directed compounds bypassing resistance to specific BCL-2 inhibition.

Discovery of novel cMET-targeting antibody Fab drug conjugates as potential treatment for solid tumors with highly expressed cMET

ABSTRACT Background Solid tumors are becoming prevalent affecting both old and young populations. Numerous solid tumors are associated with high cMET expression. The complexity of solid tumors combined with the highly interconnected nature of the cMET/HGF pathway with other cellular pathways make the pursuit of finding an effective treatment extremely challenging. The current standard of care for these malignancies is mostly small molecule-based chemotherapy. Antibody-based therapeutics as well as antibody drug conjugates are promising emerging classes against cMET-overexpressing solid tumors. Research design and methods In this study, we described the design, synthesis, in vitro and in vivo characterization of cMET-targeting Fab drug conjugates (FDCs) as an alternative therapeutic strategy. The format is comprised of a Fab conjugated to a potent cytotoxic drug via a cleavable linker employing lysine-based and cysteine-based conjugation chemistries. Results We found that the FDCs have potent anti-tumor efficacies in cancer cells with elevated overexpression of cMET. Moreover, they demonstrated a remarkable anti-tumor effect in a human gastric xenograft mouse model. Conclusions The FDC format has the potential to overcome some of the challenges presented by the other classes of therapeutics. This study highlights the promise of antibody fragment-based drug conjugate formats for the treatment of solid tumors.

PER2 binding to HSP90 enhances immune response against oral squamous cell carcinoma by inhibiting IKK/NF-κB pathway and PD-L1 expression

BackgroundProgrammed death-ligand 1 (PD-L1) contributes to the immune escape of tumor cells and is a critical target for antitumor immunotherapy. However, the molecular mechanisms regulating PD-L1 expression remain unclear, hindering...

M4IDP stimulates ROS elevation through inhibition of mevalonate pathway and pentose phosphate pathway to inhibit colon cancer cells

Maintaining redox homeostasis is an essential feature of cancer cells, and disrupting this homeostasis to cause oxidative stress and induce cell death is an important strategy in cancer therapy. M4IDP, a zoledronic acid derivative, can cause the death of human colorectal cancer cells by increasing the level of intracellular reactive oxygen species (ROS). However, its potential molecular mechanism is unclear. Our in vitro studies showed that treatment with M4IDP promoted oxidative stress in HCT116 cells, as measured by the decreased ratios of GSH/GSSG and NADPH/NADP+ and increased level of MDA. M4IDP could cause the decrease of GSH content, the increase of GSSG content, the decrease of NADPH content and pentose phosphate pathway flux, the downregulation of G6PD expression, the upregulation of unprenylated Rap1A and total expression of RhoA and CDC42. The increase of ROS and cytotoxicity induced by M4IDP could be reversed by the supplementation of NADPH, the overexpression of G6PD and the supplementation of GGOH. In vivo studies showed that M4IDP inhibited tumor growth in the human colorectal cancer xenograft mouse model, which was accompanied with a decreased [18F]FDG uptake. Collectively, these results provide evidence that M4IDP can promote oxidation in colon cancer cells by inhibiting mevalonate pathway and pentose phosphate pathway and produce therapeutic effect. This study revealed for the first time a possible mechanism of bisphosphonate-induced increase of ROS in malignant tumor cells. This is helpful for the development of new molecular therapeutic targets and can provide new ideas for the combined therapy of bisphosphonates in tumors.

Methylcrotonyl-CoA carboxylase subunit 1 (MCCA) regulates multidrug resistance in multiple myeloma

AimsThis study aimed to investigate the effect and mechanism of methylcrotonyl-CoA carboxylase subunit 1 (MCCA) on multidrug resistance in multiple myeloma (MM). Materials and methodsThe apoptosis kit and CCK-8 reagent were used to detect drug-induced cell apoptosis and viability. Immunoprecipitation, immunofluorescence staining, and protein structural simulation were used to detect the interaction between MCCA and Bad. Immunodeficient mice were injected with ARD cells and treated with bortezomib. Changes in tumor burden were recorded by bioluminescence imaging, and κ light chain content in the blood of mice was detected by enzyme-linked immunoassay. Key findingsPatients with high MCCA expression from a primary MM dataset had superior overall survival. After treatment with different anti-MM drugs, MCCA knockdown MM (MCCA-KD) cells had higher survival rates than control knockdown (CTR-KD) cells (p < 0.05). Mechanistic studies have revealed that MCCA-KD cells had dysfunctional mitochondria with decreased Bax and Bad levels and increased Bcl-xl and Mcl-1 levels. Furthermore, that MCCA and Bad demonstrated protein–protein interactions. The half-life of Bad in MCCA-KD cells is significantly shorter than that in CTR-KD cells (7.34 vs. 2.42 h, p < 0.05). In a human MM xenograft mouse model, we confirmed that MCCA-KD tumors had a poor response to anti-MM drugs in vivo. Finally, we showed that MCCA might contribute to multidrug resistance in different human cancers, particularly in solid tumors. SignificanceOur findings demonstrated a novel function of MCCA in multidrug resistance. The lack of MCCA expression promoted antiapoptotic cell signaling in MM cells.

Anti-Cancer Potential of Transiently Transfected HER2-Specific Human Mixed CAR-T and NK Cell Populations in Experimental Models: Initial Studies on Fucosylated Chondroitin Sulfate Usage for Safer Treatment.

Human epidermal growth factor receptor 2 (HER2) is overexpressed in numerous cancer cell types. Therapeutic antibodies and chimeric antigen receptors (CARs) against HER2 were developed to treat human tumors. The major limitation of anti-HER2 CAR-T lymphocyte therapy is attributable to the low HER2 expression in a wide range of normal tissues. Thus, side effects are caused by CAR lymphocyte "on-target off-tumor" reactions. We aimed to develop safer HER2-targeting CAR-based therapy. CAR constructs against HER2 tumor-associated antigen (TAA) for transient expression were delivered into target T and natural killer (NK) cells by an effective and safe non-viral transfection method via nucleofection, excluding the risk of mutations associated with viral transduction. Different in vitro end-point and real-time assays of the CAR lymphocyte antitumor cytotoxicity and in vivo human HER2-positive tumor xenograft mice model proved potent cytotoxic activity of the generated CAR-T-NK cells. Our data suggest transient expression of anti-HER2 CARs in plasmid vectors by human lymphocytes as a safer treatment for HER2-positive human cancers. We also conducted preliminary investigations to elucidate if fucosylated chondroitin sulfate may be used as a possible agent to decrease excessive cytokine production without negative impact on the CAR lymphocyte antitumor effect.

YBX1 as an oncogenic factor in T-cell acute lymphoblastic leukemia.

Y-box-binding protein 1 (YBX1), a member of the RNA-binding protein family, is a critical regulator of cell survival in various solid tumors and acute myeloid leukemia. However, the function of YBX1 in T-cell acute lymphoblastic leukemia (T-ALL) remains elusive. Here, we found that YBX1 was upregulated in patients with T-ALL, T-ALL cell lines, and NOTCH1-induced T-ALL mice. Furthermore, depletion of YBX1 dramatically reduced cell proliferation, induced cell apoptosis, and induced G0/G1 phase arrest invitro. Moreover, YBX1 depletion significantly decreased the leukemia burden in the human T-ALL xenograft and NOTCH1-induced T-ALL mice model invivo. Mechanistically, downregulation of YBX1 markedly inhibited the expression of total AKT serine/threonine kinase (AKT), p-AKT, total extracellular signal-regulated kinase (ERK), and p-ERK in T-ALL cells. Taken together, our results uncovered a critical role of YBX1 in the leukemogenesis of T-ALL, which may have great potential as a biomarker and therapeutic target in T-ALL.

Abstract A005: Regulation of HHLA2 expression in renal cell carcinoma and myeloid cells

Abstract The immune checkpoint HERV-H LTR-associating 2 (HHLA2) is expressed in many tumors, including clear cell renal cell carcinoma (ccRCC). Multiple therapeutics directed against HHLA2 or its inhibitory receptor KIR3DL3 are about to enter clinical trials in metastatic RCC; however, little is known about the regulation of HHLA2 expression. Better understanding the regulation of HHLA2 in tumor cells and within the tumor microenvironment will facilitate translation of these therapeutic agents into the clinic by assisting in patient selection and development of rational drug combinations. Using flow cytometry, immunohistochemistry (IHC), and RNA analysis, HHLA2 expression was examined in monocytes, primary nephrectomy specimens and the established ccRCC cell lines, A498 and 786-0. A panel of cytokines was screened for the induction of HHLA2 in vitro. In vivo expression of HHLA2 in A498 and 786-0 human kidney cancer xenograft mouse models was examined. Tumor cells obtained from these xenograft models (either freshly isolated or cultured ex vivo) were characterized by RNASeq and flow cytometry. Analysis of HHLA2 expression in monocytes shows that among a wide range of cytokines tested, only IL-10 and BMP4, and to a lesser extent, IL-1b and IL-6, modestly enhanced HHLA2 mRNA and protein expression. These combinations of cytokines failed to induce HHLA2 in both A498 and 786-0. HHLA2 was frequently expressed in primary ccRCC tumors by IHC. Interestingly, analysis of HHLA2 by flow cytometry in primary RCC tumors showed that HHLA2 is progressively lost over 4 weeks when cells are cultured ex vivo. Hypoxia alone did not change HHLA2 expression. While HHLA2 was not expressed on either A498 or 786O cells in vitro, both A498 and 786-0 cells expressed HHLA2 when grown as subcutaneous xenografts in NSG immunodeficient mice. A498 xenografts express significantly more HHLA2 than 786-0 xenografts. HHLA2 expression is differentially regulated in monocytes and kidney cancer epithelial cells. High concentrations of cytokines, particularly IL10, that induce HHLA2 expression in monocytes fail to upregulate HHLA2 expression in ccRCC; however, the tumor microenvironment in NSG mice significantly induced HHLA2 expression in both A498 and 786-0 xenografts. Complementary whole genome CRISPR knock out and gain-of-function screens are underway to assess by an unbiased means both nonimmune and immunologic regulators or repressors that may be responsible for inducing HHLA2 in kidney cancer in vivo. Furthermore, the A498 xenograft mouse model is an excellent humanized HHLA2 model for testing regulation of HHLA2 and HHLA2-directed therapeutics. Citation Format: Tomonari Shigemura, Nahuel Perrot, Zimo Huang, Aseman Bagheri Sheshdeh, Nourhan El Ahmar, David McDermott, Sabina Signoretti, Gordon Freeman, Kathleen Mahoney. Regulation of HHLA2 expression in renal cell carcinoma and myeloid cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A005.

Effect of a CrossMab cotargeting CD20 and HLA-DR in non-Hodgkin lymphoma

Abstract Objectives To evaluate the anti-tumor activities of CD20/HLA-DR CrossmabCH1-CL through cell and animal models. Methods Based on “knobs-into-holes” and “crossover” technology, CrossMab, targeting CD20 and HLA-DR, was constructed. A binding assay and a competitive inhibition assay were performed to confirm its specificity. The effects of CrossMab on antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity were measured. Cell apoptosis, lysosome-mediated cell death, and lysosomal permeability were quantified. In addition, the effects of CrossMab on peripheral blood leukocytes were tested. The pharmacokinetics were determined with a noncompartmental analysis model. Human malignant lymphoma xenograft models in CB17-SCID mice were established for an in-vivo efficacy study. Results The antitumor activities of CrossMab were shown both in vitro and in vivo. CrossMab exhibited strong binding to CD20 and HLA-DR at the same time in Raji cells. CrossMab also demonstrated antilymphoma effects by inducing antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Furthermore, CrossMab altered the lysosomal membrane permeability. The toxicity of CrossMab on normal peripheral blood lymphocytes (PBLs) was specific to B cells. A mouse xenograft model demonstrated the antitumor activities of CrossMab in vivo. Conclusions CrossMab exhibited an enhanced antigen recognition ability and antitumor activities in lymphoma without evident toxicity. CrossMab could be an effective immunotherapeutic strategy for non-Hodgkin lymphoma.

Multimodality PET and Near-Infrared Fluorescence Intraoperative Imaging of CEA-Positive Colorectal Cancer

PurposeMolecular imaging is a major diagnostic component for cancer management, enabling detection, staging of disease, targeting therapy, and monitoring the therapeutic response. The coordination of multimodality imaging techniques further enhances tumor localization. The development of a single agent for real-time non-invasive targeted positron emission tomography (PET) imaging and fluorescence guided surgery (FGS) will provide the next generation tool in the surgical management of cancer.ProceduresThe humanized anti-CEA M5A-IR800 “sidewinder” (M5A-IR800-SW) antibody-dye conjugate was designed with a NIR 800 nm dye incorporated into a PEGylated linker and conjugated with the metal chelate p-SCN-Bn-deferoxamine (DFO) for zirconium-89 PET imaging (89Zr, half-life 78.4 h). The dual-labeled 89Zr-DFO-M5A-SW-IR800 was evaluated for near infrared (NIR) fluorescence imaging, PET/MRI imaging, terminal tissue biodistribution, and blood clearance in a human colorectal cancer LS174T xenograft mouse model.ResultsThe 89Zr-DFO-M5A-SW-IR800 NIR fluorescence imaging showed high tumor targeting with normal liver uptake. Serial PET/MRI imaging was performed at 24 h, 48 h, and 72 h and showed tumor localization visible at 24 h that persisted throughout the experiment. However, the PET scans showed higher activity for the liver than the tumor, compared to the NIR fluorescence imaging. This difference is an important finding as it quantifies the expected difference due to the sensitivity and depth of penetration between the 2 modalities.ConclusionsThis study demonstrates the potential of a pegylated anti-CEA M5A-IR800-Sidewinder for NIR fluorescence/PET/MR multimodality imaging for intraoperative fluorescence guided surgery.

Ready vascular permeability of a near-infrared fluorescent agent ASP5354 for intraoperative ureteral identification enables imaging of carcinoma tissues

This study investigates the ability of a near-infrared fluorescence (NIRF) imaging agent, ASP5354, for in vivo fluorescence imaging of esophageal squamous cell carcinoma (ESCC) tissues. The ability of ASP5354 was evaluated using a single dose of ASP5354 or indocyanine green (ICG), which was intravenously administered to a KYSE850 human ESCC xenograft mouse model. Subsequently, in vivo NIRF images of the mouse were obtained using a clinically available camera system. ASP5354-specific NIRF signals were strongly detectable in KYSE850 carcinoma tissues immediately (30 s) following ASP5354 administration compared with normal tissues. Meanwhile, ICG could not distinguish between normal and carcinomatous tissues. To elucidate the associated imaging mechanisms, the vascular permeability of ASP5354 and ICG was investigated in rat back dermis treated with saline or histamine, which enhances vascular permeability, using in vivo NIRF imaging. ASP5354 exhibited higher vascular permeability in histamine-treated skin than in normal skin. KYSE850 carcinoma tissues can be distinguished from normal tissues based on the measurement of ASP5354-specific NIRF signals, and the mechanism that enables imaging relies on the specific and rapid leakage of ASP5354 from the capillaries into the stroma of carcinoma tissues.

Purification and functional characterization of novel human skeletal stem cell lineages.

Human skeletal stem cells (hSSCs) hold tremendous therapeutic potential for developing new clinical strategies to effectively combat congenital and age-related musculoskeletal disorders. Unfortunately, refined methodologies for the proper isolation of bona fide hSSCs and the development of functional assays that accurately recapitulate their physiology within the skeleton have been lacking. Bone marrow-derived mesenchymal stromal cells (BMSCs), commonly used to describe the source of precursors for osteoblasts, chondrocytes, adipocytes and stroma, have held great promise as the basis of various approaches for cell therapy. However, the reproducibility and clinical efficacy of these attempts have been obscured by the heterogeneous nature of BMSCs due to their isolation by plastic adherence techniques. To address these limitations, our group has refined the purity of individual progenitor populations that are encompassed by BMSCs by identifying defined populations of bona fide hSSCs and their downstream progenitors that strictly give rise to skeletally restricted cell lineages. Here, we describe an advanced flow cytometric approach that utilizes an extensive panel of eight cell surface markers to define hSSCs; bone, cartilage and stromal progenitors; and more differentiated unipotent subtypes, including an osteogenic subset and three chondroprogenitors. We provide detailed instructions for the FACS-based isolation of hSSCs from various tissue sources, in vitro and in vivo skeletogenic functional assays, human xenograft mouse models and single-cell RNA sequencing analysis. This application of hSSC isolation can be performed by any researcher with basic skills in biology and flow cytometry within 1-2 days. The downstream functional assays can be performed within a range of 1-2 months.