[Alveolar Soft Part Sarcoma-The Angiogenic Mechanism Regulated by a Fusion Gene Product].

Alveolar soft part sarcoma(ASPS)is a rare malignant tumor whose origin is unidentified, arising from deep soft tissue and affecting adolescents and young adults. ASPS is characterized by its abundant vascular network forming alveolar structures, and demonstrates frequent hematogenous metastasis. An ASPSCR1-TFE3 fusion gene derived from t(X;17)chromosome translocation is detected as a disease gene in all cases, and the ASPSCR1-TFE3 protein causes abnormal transcriptional regulation. We generated a mouse model for ASPS by introducing ASPSCR1-TFE3 into mouse embryonic mesenchymal cells. In the model, tumor angiogenesis and alveolar structures of human ASPS were reproduced, revealing pericyte-rich blood vessels and metastatic processes with pericytic encapsulation of tumor cell nests. ASPSCR1-TFE3 is frequently associated with active enhancers and super-enhancers, and angiogenesis-related enhancers were significantly diminished by the loss of ASPSCR1- TFE3. Angiogenesis-associated enhancers and important target genes, Rab27a, Sytl2, Pdgfb and Vwf were identified by epigenetic CRISPR screening. Rab27a and Sytl2 facilitates trafficking of cytoplasmic vesicles containing angiogenic factors such as Pdgfb and Vwf, resulting in pericyte-rich vascular structures in ASPS. These studies highlight the importance of the Rab27/Sytl axis as a novel drug target in cancer.

PMab-314: An Anti-Giant Panda Podoplanin Monoclonal Antibody.

The giant panda (Ailuropoda melanoleuca) is one of the important species in worldwide animal conservation. Because it is essential to understand the disease of giant panda for conservation, histopathological analyses of tissues are important to understand the pathogenesis. However, monoclonal antibodies (mAbs) against giant panda-derived proteins are limited. Podoplanin (PDPN) is an essential marker of lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. PDPN is also overexpressed in various human tumors, which are associated with poor prognosis. Here, an anti-giant panda PDPN (gpPDPN) mAb, PMab-314 (mouse IgG1, kappa) was established using the Cell-Based Immunization and Screening method. PMab-314 recognized N-terminal PA16-tagged gpPDPN-overexpressed Chinese hamster ovary-K1 cells (CHO/PA16-gpPDPN) in flow cytometry. The KD value of PMab-314 for CHO/PA16-gpPDPN was determined as 1.3 × 10-8 M. Furthermore, PMab-314 is useful for detecting gpPDPN in western blot analysis. These findings indicate that PMab-314 is a useful tool for the analyses of gpPDPN-expressed cells.

Mimicking angiogenic microenvironment of alveolar soft-part sarcoma in a microfluidic coculture vasculature chip.

Alveolar soft-part sarcoma (ASPS) is a slow-growing soft tissue sarcoma with high mortality rates that affects adolescents and young adults. ASPS resists conventional chemotherapy; thus, decades of research have elucidated pathogenic mechanisms driving the disease, particularly its angiogenic capacities. Integrated blood vessels that are rich in pericytes (PCs) and metastatic potential are distinctive of ASPS. To mimic ASPS angiogenic microenvironment, a microfluidic coculture vasculature chip has been developed as a three-dimensional (3D) spheroid composed of mouse ASPS, a layer of PCs, and endothelial cells (ECs). This ASPS-on-a-chip provided functional and morphological similarity as the in vivo mouse model to elucidate the cellular crosstalk within the tumor vasculature before metastasis. We successfully reproduce ASPS spheroid and leaky vessels representing the unique tumor vasculature to assess effective drug delivery into the core of a solid tumor. Furthermore, this ASPS angiogenesis model enabled us to investigate the role of proteins in the intracellular trafficking of bioactive signals from ASPS to PCs and ECs during angiogenesis, including Rab27a and Sytl2. The results can help to develop drugs targeting the crosstalk between ASPS and the adjacent cells in the tumoral microenvironment.

Establishment of a Novel Anti-Mouse CCR1 Monoclonal Antibody C1Mab-6.

C-C motif chemokine receptor 1 (CCR1/CD191) is a member of G-protein-coupled receptors and is expressed on myeloid cells, such as neutrophils and macrophages. Because the CCR1 signaling promotes tumor expansion in the tumor microenvironment (TME), the modification of TME is an effective strategy for cancer therapy. Although CCR1 is an attractive target for solid tumors and hematological malignancies, therapeutic agents for CCR1 have not been approved. Here, we established a novel anti-mouse CCR1 (mCCR1) monoclonal antibody (mAb), C1Mab-6 (rat IgG2b, kappa), using the Cell-Based Immunization and Screening method. Flow cytometry and Western blot analyses showed that C1Mab-6 recognizes mCCR1 specifically. The dissociation constant of C1Mab-6 for mCCR1-overexpressed Chinese hamster ovary-K1 was determined as 3.9 × 10-9 M, indicating that C1Mab-6 possesses a high affinity to mCCR1. These results suggest that C1Mab-6 could be a useful tool for targeting mCCR1 in preclinical mouse models.

A Cancer-Specific Monoclonal Antibody against HER2 Exerts Antitumor Activities in Human Breast Cancer Xenograft Models.

Monoclonal antibody (mAb)-based and/or cell-based immunotherapies provide innovative approaches to cancer treatments. However, safety concerns over targeting normal cells expressing reactive antigens still exist. Therefore, the development of cancer-specific mAbs (CasMabs) that recognize cancer-specific antigens with in vivo antitumor efficacy is required to minimize the adverse effects. We previously screened anti-human epidermal growth factor receptor 2 (HER2) mAbs and successfully established a cancer-specific anti-HER2 mAb, H2Mab-250/H2CasMab-2 (IgG1, kappa). In this study, we showed that H2Mab-250 reacted with HER2-positive breast cancer cells but did not show reactivity to normal epithelial cells in flow cytometry. In contrast, a clinically approved anti-HER2 mAb, trastuzumab, recognized both breast cancer and normal epithelial cells. We further compared the affinity, effector activation, and antitumor effect of H2Mab-250 with trastuzumab. The results showed that H2Mab-250 exerted a comparable antitumor effect with trastuzumab in the mouse xenograft models of BT-474 and SK-BR-3, although H2Mab-250 possessed a lower affinity and effector activation than trastuzumab in vitro. H2Mab-250 could contribute to the development of chimeric antigen receptor-T or antibody-drug conjugates without adverse effects for breast cancer therapy.

Suppression of Sleeping Beauty-induced Gliomagenicity in Ts1Cje Mice, a Model of Down Syndrome.

BACKGROUND/AIM: Individuals with Down syndrome (DS), attributed to triplication of human chromosome 21 (Hsa21), exhibit a reduced incidence of solid tumors. However, the prevalence of glioblastoma among individuals with DS remains a contentious issue in epidemiological studies. Therefore, this study examined the gliomagenicity in Ts1Cje mice, a murine model of DS. MATERIALS AND METHODS: We employed the Sleeping Beauty transposon system for the integration of human oncogenes into cells of the subventricular zone of neonatal mice. RESULTS: Notably, Sleeping Beauty-mediated de novo murine gliomagenesis was significantly suppressed in Ts1Cje mice compared to wild-type mice. In glioblastomas of Ts1je mice, we observed an augmented presence of M1-polarized tumor-associated macrophages and microglia, known for their anti-tumor efficacy in the early stage of tumor development. CONCLUSION: Our findings in a mouse model of DS offer novel perspectives on the diminished gliomagenicity observed in individuals with DS.

CIC-DUX4 Chromatin Profiling Reveals New Epigenetic Dependencies and Actionable Therapeutic Targets in CIC-Rearranged Sarcomas.

CIC-DUX4-rearranged sarcoma (CDS) is a rare and aggressive soft tissue tumor that occurs most frequently in young adults. The key oncogenic driver of this disease is the expression of the CIC-DUX4 fusion protein as a result of chromosomal rearrangements. CIC-DUX4 displays chromatin binding properties, and is therefore believed to function as an aberrant transcription factor. However, the chromatin remodeling events induced by CIC-DUX4 are not well understood, limiting our ability to identify new mechanism-based therapeutic strategies for these patients. Here, we generated a genome-wide profile of CIC-DUX4 DNA occupancy and associated chromatin states in human CDS cell models and primary tumors. Combining chromatin profiling, proximity ligation assays, as well as genetic and pharmacological perturbations, we show that CIC-DUX4 operates as a potent transcriptional activator at its binding sites. This property is in contrast with the repressive function of the wild-type CIC protein, and is mainly mediated through the direct interaction of CIC-DUX4 with the acetyltransferase p300. In keeping with this, we show p300 to be essential for CDS tumor cell proliferation; additionally, we find its pharmacological inhibition to significantly impact tumor growth in vitro and in vivo. Taken together, our study elucidates the mechanisms underpinning CIC-DUX4-mediated transcriptional regulation.

PMab-301: An Anti-Giraffe Podoplanin Monoclonal Antibody for Immunohistochemistry.

Immunohistochemistry staining is an essential method in pathological diagnoses. Podoplanin (PDPN) is a specific maker of alveolar epithelium, lymphatic vessels, and glomeruli. In this study, we established a novel anti-giraffe PDPN (girPDPN) mAb, PMab-301, using the Cell-Based Immunization and Screening (CBIS) method. PMab-301 (mouse IgG1, kappa) detected girPDPN in various applications, such as flow cytometry, western blot, and immunohistochemistry. PMab-301 specifically stained type-I alveolar cells using formalin-fixed paraffin-embedded giraffe lung tissues. Our findings suggest the potential usefulness of PMab-301 for the pathophysiological analyses of giraffe tissues.