Allografts In Mice Research Articles

Gracillin exerts anti-melanoma effects in vitro and in vivo: role of DNA damage, apoptosis and autophagy

BackgroundMelanoma is an aggressive cancer. Gracillin has been reported to treat various types of cancer, such as colorectal and lung cancer. However, there is a paucity of research on the anti-melanoma effects of gracillin. PurposeThe aim of this study was to assess the anti-melanoma effects and mechanisms of action of gracillin in vitro and in vivo. MethodsCell viability was detected using MTT and crystal violet staining assays. Cell proliferation was examined by EdU staining assays. Cell cycle arrest and apoptosis were analyzed by flow cytometry. Autophagic flux was monitored under a confocal microscope. Protein levels were determined by immunoblotting. LY294002 and rapamycin (Rapa) were used to determine the involvement of PI3K/AKT/mTOR signaling in gracillin-mediated autophagy. Signal transducer and activator of transcription 3 (STAT3) was overactivated to explore the contribution of the STAT3 signaling pathway in the anti-melanoma effects of gracillin. A B16F10 allograft mouse model was developed to evaluate the anti-melanoma effects of gracillin in vivo. ResultsWe demonstrated that in melanoma cells, gracillin inhibited proliferation, induced G0/G1 phase cell cycle arrest, evoked apoptosis, and triggered autophagic cell death. Gracillin induced DNA damage in melanoma cells. Moreover, it suppressed the phosphorylation/activation of PI3K, AKT, mTOR, and 4E-BP1 in melanoma cells. Inhibiting PI3K/AKT and mTOR activity using LY294002 and Rapa, respectively, increased the protein level of LC3B-II in gracillin-treated melanoma cells. Furthermore, gracillin downregulated the protein levels of p-JAK2 (Tyr1007/1008), p-Src (Tyr416), and p-STAT3 (Tyr705) in melanoma cells. Over-expression of STAT3 in A375 cells significantly mitigated the cytotoxic and apoptotic effects of gracillin. In vivo studies showed that gracillin (1 mg/kg or 8 mg/kg, administered intraperitoneally for 16 consecutive days) suppressed B16F10 tumor growth and Src/STAT3 and AKT/mTOR signaling in tumors. No overt toxicity was observed in mice. ConclusionInduction of DNA damage, inhibition of PI3K/AKT/mTOR signaling and suppression of STAT3 signaling are involved in gracillin-mediated cell cycle arrest, autophagic cell death and apoptosis, respectively, in melanoma cells. These findings provide novel insights into the anti-melanoma molecular mechanisms of gracillin, and suggest a potential role of gracillin in melanoma management.

Excessive HSP70/TLR2 activation leads to remodeling of the tumor immune microenvironment to resist chemotherapy sensitivity of mFOLFOX in colorectal cancer

For locally advanced colorectal cancer (CRC), neoadjuvant chemoradiotherapy (nCRT) followed by total mesorectal excision or complete mesocolic excision is the standard therapeutic strategy, which is key to patient survival. Involvement of the tumor immune microenvironment is a factor that regulates tumor progression and sensitivity to nCRT in CRC. In this study, we aimed to identify the effect of heat-shock protein 70 (HSP70)/toll-like receptor-2 (TLR-2) on mFOLFOX sensitization for CRC. A total of 22 patients with advanced CRC who had received neoadjuvant mFOLFOX were enrolled and classified into the mFOLFOX-insensitive or -sensitive group, according to the tumor regression grade. The abundance of immune infiltrates was significantly higher in the post-operative pathological specimens of the mFOLFOX-insensitive group, as compared to those of the mFOLFOX-sensitive group. After transcriptome sequencing, differentially expressed genes between the two groups were annotated to inflammatory and immune responses using Gene Ontology (GO) analysis, and the TLR signaling pathway was analyzed using Kyoto Encyclopedia of Genes and Genomes pathway analysis. Significantly higher expression levels of HSP60, HSP70, HSP90, and TLR-2 in the mFOLFOX-insensitive group were detected using immunofluorescence assays. TIMER2.0 platform was introduced to further narrow the scope of HSP70 (HSPA6 or HSPA7) and TLR-2, which exhibited positive correlations with dendritic cells, Tregs, or CD4+ T cells and negative correlations with CD3+ or CD8+ T cells, implying that HSP70/TLR-2 activation mediates immunosuppressive cells to counteract CD8+ T cells, which may be a novel target of CRC treatment. A promising synergistic effect of mFOLFOX combined with a TLR-2 inhibitor was observed in vivo in mouse allograft models, which could be partly rescued by recombinant HSP70 protein. Immunohistochemical staining of allografts and immunofluorescence assays of clinical specimens corroborated the regulatory effects of the immune microenvironment. In summary, HSP70/TLR-2 activation can regulate the tumor immune microenvironment of CRC and further remodel its sensitivity to mFOLFOX. However, the specific mechanisms remain unclear and require further investigation. This study is expected to provide a new direction for the clinical treatment of CRC.

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, immune cells, vessels, nerves, and extracellular matrix components. Over the years, different mouse models of PDAC have been developed to address the challenges posed by its progression, metastatic potential, and phenotypic heterogeneity. Immunocompetent mouse orthotopic allografts of PDAC have shown good promise owing to their fast and reproducible tumor progression in comparison to genetically engineered mouse models. Moreover, combined with their ability to mimic the biological features observed in autochthonous PDAC, cell line-based orthotopic allograft mouse models enable large-scale in vivo experiments. Thus, these models are widely used in preclinical studies for rapid genotype-phenotype and drug-response analyses. The aim of this protocol is to provide a reproducible and robust approach to successfully inject primary mouse PDAC cell cultures into the pancreas of syngeneic recipient mice. In addition to the technical details, important information is given that must be considered before performing these experiments.

Celastrol inhibits lung cancer growth by triggering histone acetylation and acting synergically with HDAC inhibitors

Alterations in histone modification have been linked to cancer development and progression. Celastrol, a Chinese herbal compound, shows potent anti-tumor effects through multiple signaling pathways. However, the involvement of histone modifications in this process has not yet been illustrated. In this study, barcode sequencing of a eukaryotic genome-wide deletion library revealed that histone modifications, especially histone acetylation associated with the NuA4 histone acetyltransferase complex, were involved in the anti-proliferation actions of celastrol. The essential roles of histone modification were verified by celastrol sensitivity tests in cells lacking specific genes, such as genes encoding the subunits of the NuA4 and Swr1 complex. The combination of celastrol and histone deacetylase inhibitors (HDACi), rather than the combination of celastrol and histone acetyltransferase inhibitors, synergistically suppressed cancer cell proliferation. In addition to upregulating H4K16 acetylation (H4K16ac), celastrol regulates H3K4 tri-methylation and H3S10 phosphorylation. Celastrol treatment significantly enhanced the suppressive effects of HDACi on lung cancer cell allografts in mice, with significant H4K16ac upregulation, indicating that a combination of celastrol and HDACi is a potential novel therapeutic approach for patients with lung cancer.

64 (PB054) - BDTX-1535, a fourth generation EGFR inhibitor, targeting intrinsic and acquired resistance mutations in NSCLC

Background: The classical mutations of the epidermal growth factor receptor (EGFR) in NSCLC are the exon 19 del and exon 21 (L858R) mutations. The 3rd generation EGFR tyrosine kinase inhibitor (TKI), osimertinib, has become the first-line treatment of choice for NSCLC patients with these mutations. Resistance to 3rd generation TKIs can be driven by gain of secondary EGFR alterations, e.g., substitution at cysteine-797 to serine (C797S). Real world evidence shows that in addition to C797S, other EGFR alterations are also acquired upon osimertinib treatment, including kinase domain mutations (e.g., S768I), extracellular domain alterations (e.g., EGFRvIII, A289X), and EGFR amplification. In addition to the classical mutations, NSCLC tumors express a wide spectrum of primary kinase domain mutations including G719X in exon 18, S768I in exon 20, and L861Q in exon 21, which confer intrinsic resistance to approved TKIs. NCCN guidelines recommend the use of afatinib or osimertinib, however, there remains a need for an EGFR TKI that targets these mutations with high potency, good tolerability, and CNS penetration. Our MAP platform allowed us to select MasterKey inhibitor BDTX-1535, a 4th-generation CNS penetrant EGFR TKI, that potently and selectively targets multiple intrinsic and acquired resistance EGFR alterations. Materials and Methods: BDTX-1535 preclinical exposure was evaluated across species, and PK and Kpuu values were calculated in brain and plasma. Antitumor activity was assessed across a broad range of mouse PDX and allograft models. Results: BDTX-1535 is a potent and selective, CNS penetrant, wild type sparing, irreversible EGFR TKI targeting Exon 18–21 alterations which are associated with intrinsic or acquired resistance to 3rd generation EGFR inhibitors. These include a broad range of kinase domain EGFR mutations (e.g., C797S, L718Q, G724S, S768I), extracellular domain alterations (e.g., EGFRvIII, A289X), and EGFR amplification. Mouse xenograft and allograft studies showed that BDTX-1535 consistently achieves regression of tumors carrying these mutations at well tolerated doses without significant EGFR wild type associated toxicities. BDTX-1535 is orally bioavailable with a CNS Kpuu of 0.55 and 0.48 in rat and dog, respectively, and active in an intracranial PDX model. Conclusions: BDTX-1535 is a 4th generation CNS penetrant EGFR TKI discovered using the MAP platform which allows MasterKey targeting of EGFR alterations associated with intrinsic or acquired resistance to 3rd generation EGFR TKIs, while sparing wild type EGFR. This broad spectrum coverage of resistance mutations in conjunction with CNS penetration properties allows BDTX-1535 to address a key unmet medical need in EGFR mutant lung cancer. BDTX-1535 is currently under phase I clinical investigation (NCT05256290). Conflict of interest: Ownership: Black Diamond Therapeutics

66 (PB056) - Anti-tumor activity of BDTX-1535, an irreversible CNS penetrant inhibitor of multiple EGFR extracellular domain alterations, in preclinical glioblastoma models

Background: EGFR genetic alterations, including mutation and amplification, are very common in glioblastoma multiforme (GBM), however, no targeted therapy has been approved for this disease. EGFR oncogenic mutations in GBM promote EGFR oncogenic covalent homodimer formation which leads to paradoxical activation and renders reversible inhibitors ineffective. An effective EGFR inhibitor in GBM must meet four conditions:1Highly potent and selective against a broad range of extracellular domain mutations and amplified EGFR,2Wild-type EGFR sparing,3Irreversible binding to circumvent paradoxical activation,4High CNS penetration. Our MAP discovery platform led to the identification of MasterKey inhibitor BDTX-1535, an irreversible 4th-generation CNS penetrant EGFR tyrosine kinase inhibitor (TKI) that potently and selectively targets a broad range of EGFR alterations in GBM. Materials and Methods: BDTX-1535 preclinical exposure was evaluated in multiple species in plasma and brain. Antitumor activity was assessed across a broad range of mouse PDX and allograft models, including an intracranial PDX. 2540 tumors were sequenced with the Tempus xT NGS assay. Mutational frequencies and co-expression status of oncogenic EGFR variants were assessed. The relationship between treatment history and oncogenic EGFR mutational frequency was analyzed. Results: BDTX-1535 is a small molecule that irreversibly inhibits a spectrum of EGFR alterations expressed in GBM together with EGFR amplification, while sparing inhibition of normally expressed wild type EGFR. BDTX-1535 avoids efficacy limiting paradoxical activation that can occur with reversible EGFR inhibitors. BDTX-1535 exhibits good oral bioavailability and a CNS Kpuu of 0.55 and 0.48 in rat and dog, respectively, comparing favorably with other CNS penetrant TKIs. Tumor regression and survival advantage were observed across all tumor models with EGFR amplification or mutation, including in an intracranial PDX model. EGFR mutations and amplification are commonly expressed in GBM. Consistent with published data, EGFR mutations, variants, and copy number gain were frequently co-expressed. For example, among tumors expressing EGFRvIII, ∼95% co-expressed at least one other variant, and among tumors expressing EGFRvVI, ∼45% co-expressed at least one other variant. Within a subset of GBM patients with longitudinal sequencing data, oncogenic EGFR mutations were expressed and persisted following front-line treatment. Conclusions: BDTX-1535 is a potent, CNS penetrant, irreversible small molecule EGFR inhibitor that shows efficacy against a broad range of extracellular domain mutations and EGFR amplification found in GBM patients. RWE findings demonstrate a heterogeneous EGFR mutational profile in GBM patients, with persistent prevalence over time. BDTX-1535 is currently in a phase I clinical study (NCT05256290). Conflict of interest: Ownership: Black Diamond Therapeutics Tempus Labs

CXCR4 and CXCR7 signaling promotes tumor progression and obesity-associated epithelial-mesenchymal transition in prostate cancer cells.

Obesity is associated with increased prostate cancer (PCa) progression and higher mortality, however, the mechanism(s) remain still unclear. Here, we investigated signaling by the ASC-secreted chemokine CXCL12 in a mouse allograft model of PCa and in HiMyc mice in the context of diet-induced obesity. Treatment of mice with CXCR4 antagonist inhibited CXCL12-induced signaling pathways, tumor growth and EMT in HMVP2 allograft tumors. Similar results were obtained following prostate epithelium-specific deletion of CXCR4 in HiMyc mice. We also show that CXCR4 signaling regulates expression of JMJD2A histone demethylase and histone methylation which is modulated by AMD3100. Importantly, treatment with a CXCR7 antagonist also inhibited allograft tumor growth and EMT. The current results demonstrate that both CXCR4 and CXCR7 play an important role in cancer progression and establish CXCL12 signaling pathways, activated in obesity, as potential targets for PCa intervention. In addition, other factors secreted by ASCs, may also contribute to cancer aggressiveness in obesity.

Host-versus-commensal immune responses participate in the rejection of colonized solid organ transplants.

Solid organ transplantation is the preferred treatment for end-stage organ failure. Although transplant recipients take life-long immunosuppressive drugs, a substantial percentage of them still reject their allografts. Strikingly, barrier organs colonized with microbiota have significantly shorter half-lives than non-barrier transplanted organs, even in immunosuppressed hosts. We previously demonstrated that skin allografts monocolonized with the common human commensal Staphylococcus epidermidis (S.epi) are rejected faster than germ-free (GF) allografts in mice because the presence of S.epi augments the effector alloimmune response locally in the graft. Here, we tested whether host immune responses against graft-resident commensal microbes, including S.epi, can damage colonized grafts independently from the alloresponse. Naive hosts mounted an anticommensal T cell response to colonized, but not GF, syngeneic skin grafts. Whereas naive antigraft commensal T cells modestly damaged colonized syngeneic skin grafts, hosts with prior anticommensal T cell memory mounted a post-transplant immune response against graft-resident commensals that significantly damaged colonized, syngeneic skin grafts. Importantly, allograft recipients harboring this host-versus-commensal immune response resisted immunosuppression. The dual effects of host-versus-commensal and host-versus-allograft responses may partially explain why colonized organs have poorer outcomes than sterile organs in the clinic.

315.14: The Stem Cell Mobilizator Plerixafor Reduces Transplant Vasculopathy in a Murine Aortic Allograft Model

Purpose: Effector and regulatory T cells (Tregs) are main cell populations in the context of cardiac allograft vasculopathy (CAV). The immunostimulant plerixafor mobilizes hematopoietic stem cells and may boost T cell. Therefore, the aim of this study was to evaluate if a single or repeated application as well as continuous treatment with plerixafor increases peripheral bone marrow derived stem cells including Tregs leading to tolerance induction and thus reducing chronic rejection in a mouse allograft transplant model. Methods: Fully allogeneic mouse aortas from C57BL/6 mice (H2b) were abdominally transplanted into CBA mice (H2k). The continuous plerixafor application [1mg/kg/d] was carried out for 14 days using implanted small osmotic pumps. Single dose (s.c.) injection of 1mg/kg/d or 5mg/kg/d were done at day 1 after transplantation (Tx.) and pulsed injections [1mg/kg/d] were conducted on days 1, 7, 14, and 21 after Tx. The control allograft group received vehicle loaded osmotic pumps. Stem cell mobilization was monitored by FACS. Recipients were sacrificed on day 14 for intragraft gene expression analysis or on day 30 for histological measurements. Results: Murine aortic grafts with pulsed plerixafor injections showed significantly reduced neointima proliferation compared to control allografts (33.65%±8.84% vs.53.13% ±12.41%; p<0.05). The single shot and continuous treatment groups exhibited no improvement concerning neointima formation vs. the untreated group. First FACS analysis revealed significantly less hematopoietic stem cells (HSC) in the bone marrow of plerixafor treated mice vs. the control at day 14 after Tx. Though, there were significantly more HSCs in the peripheral blood on day 30 after Tx, with the pulsed injection even doubling HSCs [0.0152%±0.008; p < 0.005 (pulsed); 0.0046%±0.002%; p < 0.01 (pump); 0.0076%±0.001%; p < 0.01 (single dose of 1mg/kg); 0.0039%±0.0017%; p < 0.1 (single dose of 5mg/kg) vs. 0.0018%±0.0016% (control)]. Intragraft gene expression results showed clearly reduced IFNγ expression and a significantly increase of IL-4, IL-10 and TGFβ. Conclusion: The data suggests that pulsed, continued and single dose application of plerixafor leads to potent stem cell mobilization and hereby the repeated treatment with plerixafor reduces neointima formation in a mouse aortic transplant model. Manfred-Roth Stiftung. Forschungsstiftung Medizin, FAU Erlangen-Nürnberg.

AQP9 transports lactate in tumor-associated macrophages to stimulate an M2-like polarization that promotes colon cancer progression

Macrophages play a major role in the immune defense against pathogenic factors; however, they can lead to tumor exacerbation and metastasis, as the tumor microenvironment (TME) polarizes tumor-associated macrophages (TAMs) into the M2 subtype. Lactate, a metabolite produced by carcinoma cells at high concentrations in the TME, induces an M2-polarization in macrophages, which ultimately leads to the secretion of factors, such as vascular endothelial growth factor (VEGF), and promotes tumor progression. However, the effect of TAM lactate import on tumor progression has not been fully elucidated. Aquaporin 9 (AQP9) is a transporter of water and glycerol expressed in macrophages. Here, we used a tumor allograft mouse model to show that AQP9 knockout (AQP9−/−) mice were more resistant against tumor cell growth and exhibited a suppressive M2-like polarization in tumor tissue than wild-type mice. Moreover, we discovered that the primary bone marrow-derived macrophages from AQP9−/− mice were less sensitive to lactate stimulation and exhibited reduced M2-like polarization as well as decreased VEGF production. To further investigate the role of AQP9 in macrophage polarization, we overexpressed AQP9 in Chinese hamster ovary cells and found that AQP9 functioned in lactate import. In contrast, primary AQP9−/− macrophages and AQP9 knockdown RAW264.7 cells exhibited a reduced lactate transport rate, suggesting the involvement of AQP9 in lactate transport in macrophages. Together, our results reveal the mechanism by which the TME modifies the polarization and function of tumor-infiltrating macrophages via AQP9 transport function.

Anti-tumor effect of aquaporin 3 monoclonal antibody on syngeneic mouse tumor model

Aquaporin-3 (AQP3), a water channel protein, has been found to be involved in cancer progression via water and small molecule transport function. However, drug development targeting AQP3 has not yet begun.Here, we showed that a recently established anti-AQP3 monoclonal antibody (mAb) suppresses tumor growth in allograft mouse colorectal tumor models produced using CT26 or MC38 cancer cells. Administration of the anti-AQP3 mAb to BALB/c mice with transplanted CT26 cells increased the M1/M2 ratio of tumor-associated macrophages (TAM) and improved the mitochondrial function of T cells in the tumor microenvironment (TME). Administration of anti-AQP3 mAb also restored the TAM-induced decrease in T cell proliferation. Macrophage depletion in wild-type mice counteracted the antitumor effect of anti-AQP3 mAb in the mouse tumor model, suggesting that one of the primary targets of anti-AQP3 mAb is macrophages. In in vitro studies using mice bone marrow monocytes and human monocyte THP-1 cells, anti-AQP3 mAb attenuated carcinoma cell-mediated polarization of monocytes into M2-like TAMs.These data suggest that anti-AQP3 mAb suppresses tumor growth by attenuating immunosuppressive M2-like TAMs, which in turn maintains the antitumor function of T cells in the TME. Thus, the anti-AQP3 mAb is a potential cancer therapy that functions by targeting TAMs.

ZBTB20 regulates cardiac allograft rejection through NFкB-mediated inflammation in mouse heart transplantation

Allograft rejection is a major obstacle for the long-term survival of heart transplantation (Htx) patients. The cardiac allograft rejection requires the activation of macrophages and effector T cells. In this study, we explored the role of zinc-finger and BTB domain containing protein 20 (ZBTB20) in the regulation of heart allograft rejection. Flow cytometry analysis of the spleen cells from mice undergoing an acute cardiac rejection revealed that the ZBTB20 protein expression was upregulated in both T and B cells(n = 4，P < 0.01). In addition, ZBTB20 gene knockdown significantly prolonged the survival of heart allografts in mice(n = 4，P < 0.01). Lack of ZBTB20 increased the expression of Foxp3 and limited the response of T helper 1 (Th1) cells(n = 4，P < 0.01). The ZBTB20-related regulation occurred through the activation of the NFкB pathway. In conclusion, our data suggest that ZBTB20 is involved in the regulation of T cells involved in acute heart allograft rejection. Hence, downregulation of ZBTB20 expression may inhibit T cells to prolong heart transplant survival.

Growth hormone receptor antagonism downregulates ATP-binding cassette transporters contributing to improved drug efficacy against melanoma and hepatocarcinoma in vivo

Knockdown of GH receptor (GHR) in melanoma cells in vitro downregulates ATP-binding cassette-containing (ABC) transporters and sensitizes them to anti-cancer drug treatments. Here we aimed to determine whether a GHR antagonist (GHRA) could control cancer growth by sensitizing tumors to therapy through downregulation of ABC transporters in vivo. We intradermally inoculated Fluc-B16-F10 mouse melanoma cells into GHA mice, transgenic for a GHR antagonist (GHRA), and observed a marked reduction in tumor size, mass and tumoral GH signaling. Moreover, constitutive GHRA production in the transgenic mice significantly improved the response to cisplatin treatment by suppressing expression of multiple ABC transporters and sensitizing the tumors to the drug. We confirmed that presence of a GHRA and not a mere absence of GH is essential for this chemo-sensitizing effect using Fluc-B16-F10 allografts in GH knockout (GHKO) mice, where tumor growth was reduced relative to that in GH-sufficient controls but did not sensitize the tumor to cisplatin. We extended our investigation to hepatocellular carcinoma (HCC) using human HCC cells in vitro and a syngeneic mouse model of HCC with Hepa1-6 allografts in GHA mice. Gene expression analyses and drug-efflux assays confirm that blocking GH significantly suppresses the levels of ABC transporters and improves the efficacy of sorafenib towards almost complete tumor clearance. Human patient data for melanoma and HCC show that GHR RNA levels correlate with ABC transporter expression. Collectively, our results validate in vivo that combination of a GHRA with currently available anti-cancer therapies can be effective in attacking cancer drug resistance.

Enhancement of tumor immunogenicity by the introduction of non- proteinogenic amino acid azetidine-2-carboxylic acid

ABSTRACT Despite the clinical success in the treatment of several types of cancers, the immune checkpoint inhibitors (ICIs) show limited response rates in cancers with low tumor mutational burden (TMB) and antigenicity. Here, we aim to enhance tumor antigenicity at the protein translation level by using non-proteinogenic amino acids (NPAs) that cause regional mistranslation and mutated proteins. We utilized proline analogue azetidine-2-carboxylic acid (AZA), which can be discharged into proline tRNA by prolyl-tRNA synthetase, leading to the generation of a proportion of mutated proteins with proline residues substituted with Aze in tumor cells undergoing active protein synthesis. To specifically produce mutated proteins in tumor cells, the anti-Cd44 antibody-coated liposome nanoparticles (NPs) were used to deliver Aze specifically into the breast cancer cells. The Aze delivered by NPs can be incorporated into proteins in the 4T1 tumor allografts in mice, resulting in the activation of cellular immune responses and hence the significant inhibition of the growth of 4T1 allografts and the pulmonary metastasis, eventually prolonging the survival of tumor-bearing mice. Interestingly, Aze increases the response of 4T1 breast cancer allografts to anti-PD1 antibody treatment, suggesting Aze is able to sensitize tumors to the ICIs treatment in the immunotherapy of tumors.

Exogenous iron impairs the anti-cancer effect of ascorbic acid both in vitro and in vivo

IntroductionThe anti-cancer effect of high concentrations of ascorbic acid (AA) has been well established while its underlying mechanisms remain unclear. The association between iron and AA has attracted great attention but was still controversial due to the complicated roles of iron in tumors. ObjectivesOur study aims to explore the anti-cancer mechanisms of AA and the interaction between AA and iron in cancer. MethodsThe MTT and ATP assays were used to evaluate the cytotoxicity of AA. Reactive oxygen species (ROS) generation, calcium (Ca2+), and lipid peroxidation were monitored with flow cytometry. Mitochondrial dysfunction was assessed by mitochondrial membrane potential (MMP) detection with JC-1 or tetramethylrhodamine methyl ester (TMRM) staining. Mitochondrial swelling was monitored with MitoTracker Green probe. FeSO4 (Fe2+), FeCl3 (Fe3+), Ferric ammonium citrate (Fe3+), hemin chloride (Fe3+) were used as an iron donor to investigate the effects of iron on AA’s anti-tumor activity. The in vivo effects of AA and iron were analyzed in xenograft zebrafish and allograft mouse models. ResultsHigh concentrations of AA exhibited cytotoxicity in a panel of cancer cells. AA triggered ROS-dependent non-apoptotic cell death. AA-induced cell death was essentially mediated by the accumulated intracellular Ca2+, which was partly originated from endoplasmic reticulum (ER). Surprisingly, exogenous iron could significantly reverse AA-induced ROS generation, Ca2+ overloaded, and cell death. Especially, the iron supplements significantly impaired the in vivo anti-tumor activity of AA. ConclusionsOur study elucidated the protective roles of iron in ROS/Ca2+ mediated necrosis triggered by AA both in vitro and in vivo, which might shed novel insight into the anti-cancer mechanisms and provide clinical application strategies for AA in cancer treatment.

Therapeutic effect of multiple functional minicircle vector encoding anti-CD25/IL-10/CXCR3 in allograft rejection model.

Background/AimsWe previously proposed minicircle vector technology as the potential platform for the development and production of new biologics. In this study, we have designed a novel target molecule for the treatment of allograft rejection and evaluated its feasibility as the therapeutic agent in this disease using the minicircle vector system.MethodsWe engineered vectors to carry cassette sequences for anti-CD25, interleukin-10 (IL-10), and C-X-C motif chemokine receptor 3 (CXCR3) fusion protein, and then isolated minicircle vectors from the parent vectors. We verified the substantial production of anti-CD25/IL-10/CXCR3 fusion protein from minicircles and their duration in HEK293T cells and mice models. We also evaluated whether minicircle-derived anti-CD25/IL-10/CXCR3 has therapeutic effects in a skin allograft in mice model.ResultsWe confirmed the production of anti-CD25/IL-10/CXCR3 from minicircle by its significant availability in cells transfected with the minicircle and in its conditioned media. After a single injection of minicircle by hydrodynamic injection via mouse tail vein, luminescence or red fluorescence was maintained until 40 days in the liver tissue, suggesting the production of anti-CD25/IL-10/CXCR3 protein from minicircles via protein synthesis machinery in the liver. Mice treated with the minicircle encoding anti-CD25/IL-10/CXCR3 showed prolonged skin allograft survival times accompanied by improved immunologic regulation e.g., reduction of the lymphocyte population of Th1, Th2, and Th17 and an induction of regulatory T cells.ConclusionsThese findings implied that self-generated anti-CD25/IL-10/CXCR3 protein drug by minicircle technology is functionally active and relevant for reducing allograft rejection. The minicircle vector system may be useful for developing new biological drugs, avoiding manufacturing or practical problems.

Abstract 2743: Tumor growth modeling and its applications in preclinical pharmacology studies to improve translatability of animal models

Abstract Successful oncology drug development hinges on proper use of preclinical xenograft and allograft mouse models that faithfully reflect histopathology and genomic features of patient tumors and exhibit clinically similar drug response. For in vivo pharmacology studies, tumor-bearing mice are treated by drugs and efficacy is monitored by continuingly measuring tumor volumes over weeks or even months, resulting in mouse-specific tumor growth data. In our previous study, we used linear mixed model (LMM) to fit tumor growth data and demonstrated its superiority to simple readouts, such as tumor growth inhibition (TGI), in discovering biomarkers and exploring mechanism of action for drugs. Here we extended the study to more parametric statistical models on an expanded dataset with over 100 thousand tumor growth curves. Six parametric models (exponential, exponential square, Gompertz, Logistic, Monomolecular, and von Bertalanffy) were fit to tumor growth curves within a single-treatment group, by the least square method after variance stabilization transformation. Best models were selected by Akaike information criterion (AIC)-based confidence set. In the Irinotecan case study, logarithmic transformation of tumor volume converts exponential or exponential square models to mixed-effects linear models that integrate random effect of mouse difference within a treatment group, and are used to identify predictive biomarkers for drug efficacy. We compared three variance stabilization transformations and found that log transformation of tumor volume is appropriate for majority of tumor growth curves. In log transformed data, exponential or exponential square model can satisfactorily fit nearly all tumor growth curves, as 96.1% of best model confidence sets contain exponential or exponential square model, followed by von Bertalanffy (92.5%), Gompertz (92.1%), Logistic (90.5%), and Monomolecular (58.9%). The percentage of studies, in which all treatment groups can be best fit by exponential or exponential square model, is 91.3% (82.4% for von Bertalanffy, 81.4% for Gompertz, 79.1% for Logistic, 37.3% for Monomolecular). Thus, exponential or exponential square model can be applied to most in vivo mouse studies, and should be preferred for their mathematical simplicity and interpretability. We further demonstrated the utility of the modeling method on a 16-PDX-model study for Irinotecan to identify biological processes and pathways related to its mechanism of action. Citation Format: Huajun Zhou, Binchen Mao, Sheng Guo. Tumor growth modeling and its applications in preclinical pharmacology studies to improve translatability of animal models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2743.

Abstract 943: Modeling and dissecting of esophageal squamous cell carcinoma initiation

Abstract Esophageal cancer is the 7th common cancer with the 6th highest cancer mortality rate worldwide. The 5-year survival rate of esophageal cancer is 10 ~ 25%. Esophageal cancer is mainly divided into esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma. ESCC accounts for 80% of all esophageal cancer cases and shows a poor prognosis due to a lack of symptoms at early stages. Given that early diagnosis of ESCC may lead to better outcomes, understanding the biology of ESCC initiation is imperative. To recapitulate the early events of ESCC, we employed the murine esophageal organoid (EO) system recently developed. Based on in silico analyses, we selected nine candidate genes (TP53, CDKN2A, NOTCH1, NOTCH3, KMT2D, KMT2C, FAT1, FAT4, and AJUBA) frequently mutated in ESCC patients. The EOs were then genetically manipulated with the combinatorial ablation of nine genes by CRISPR/Cas9 system. The EOs carrying PN (Trp53 and Notch1 KO), PC (Trp53 and Cdkn2a KO), and PCN (Trp53, Cdkn2a, and Notch1 KO) showed hyperplastic and neoplastic properties with impaired squamous cell differentiation and organoid integrity, compared to the wild-type (WT) EOs. Single-cell RNA-seq (scRNA-seq) analyses of WT, PC, PN, and PCN organoids showed the highest enrichment of ESCC cancer stem cell markers in PCN organoids. Unlike the cell lineage in WT EOs, neoplastic EOs (PC, PN, and PCN) exhibited distinct cell lineage along with multiple root cell clusters. While WT EOs failed to become neoplastic, PN, PC, and PCN EOs displayed the transforming activity in vitro and in vivo xenograft transplantation assays. Intriguingly, in allograft mouse models, while PN organoids-derived cells barely developed tumors, PC and PCN organoids-derived cells developed tumors with marked downregulation of genes related to antigen processing and presentation. These results suggest that combinatorial loss of P53, CDKN2A, and NOTCH1 play a pivotal role in ESCC neoplasia, stemness, and immune checkpoint, unveiling the biology of ESCC initiation. Citation Format: Kyung Pil Ko, Gengyi Zou, Yuanjian Huang, Bongjun Kim, Jie Zhang, Sohee Jun, Jae-Il Park. Modeling and dissecting of esophageal squamous cell carcinoma initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 943.

Abstract 1573: Paradoxical roles of peritumoral myofibroblasts in intrahepatic cholangiocarcinoma growth and metastasis

Abstract Background and Rationale: Intrahepatic cholangiocarcinoma (iCCA) is an aggressive type of primary liver cancer characterized by its highly desmoplastic stroma. Compared to the ample knowledge on the protumorigenic functions of iCCA intratumoral myofibroblasts (iMFs), little is known about the activation status of MFs in the tumor-surrounding liver (peritumoral myofibroblasts, pMFs) and their potential role in iCCA development. Results: We examined MF activation in both patient iCCA tumors and an iCCA orthotopic allograft mouse model. We found there was a significant, tumor-induced activation and accumulation of pMFs at the tumor border. Using an in vitro spheroid coculture system, we confirmed that iCCA-liver interaction induced rapid pMF activation at their interface. We found that MFs mixed within tumor spheroids as iMFs had a pro-proliferative effect on iCCA tumor cells similar to the previous reports. However, when the same MFs were seeded around iCCA spheroids as pMFs, they exerted a strong suppressive effect on tumor cells growth. Interestingly, prolonged iCCA-pMF coculture elicited marked tumor cell invasion and dissemination which did not occur in iCCA-iMF coculture. We found vascular cell adhesion protein 1 (Vcam1), a glycoprotein that has been implicated in solid tumor metastasis, was part of the dynamic regulation of tumor cell behaviors by iCCA-pMF interaction. Vcam1 was upregulated in the tumor cells at the early, growth-suppressing phase of iCCA-pMF interaction both in vitro and in vivo, and was then downregulated in tumor cells when they started to disseminate. Systemic blocking of Vcam1 activity in iCCA allograft mouse models using a Vcam1 neutralizing antibody slowed the primary tumor growth but promoted tumor cell dissemination. Conclusion: Our data suggest that pMFs are beyond simple pro- or anti-tumorigenic in iCCA development. iCCA development induces rapid activation and accumulation of pMFs at the tumor border which suppress tumor growth at early stage but eventually elicit tumor cell dissemination at late stage. Citation Format: Cheng Tian, Liyuan Li, Yizhen Li, Wentao Yang, Li Fan, Anthony Brown, Michelle Morrison, Jun J. Yang, Evan S. Glazer, Liqin Zhu. Paradoxical roles of peritumoral myofibroblasts in intrahepatic cholangiocarcinoma growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1573.

Abstract 5687: CDC7 inhibitor-induced replication stress generates inflamed aneuploid cells to sensitize immune checkpoint inhibitors

Abstract Background: The serine/threonine kinase, cell division cycle 7 (CDC7), is critical for initiating DNA replication. A highly specific CDC7 inhibitor, TAK-931, was developed as a next-generation of replication stress (RS) inducer. This study preclinically investigated the novel aspects of TAK-931 on antitumor efficacy and immunity for evaluating the therapeutic potential of TAK-931 in combination with immune checkpoint inhibitors (ICIs). Methods: TAK-931-treated cells were subjected to cell growth assay, senescence-associated galactosidase (SA-β-gal) activity assay, transcriptome analysis (RNA-seq), and single-cell RNA-seq (scRNA-seq). The flowcytometry (FCM)-based immune profiling panel studies in J558 allograft syngeneic mouse models were performed. In vivo efficacy studies in J558 allograft models in combination with anti-mPD-1, anti-mPD-L1, and anti-mCTLA-4 antibodies were performed. Results: TAK-931 intensively induced RS, generating senescence-associated secretory phenotype (SASP) aneuploid cells, which highly expressed inflammatory cytokines and chemokines. In the transcriptome analyses, the inflammatory cytokine and chemokine hallmarks were significantly and intensively enriched in the TAK-931-induced aneuploid cells; five out of the top six enriched hallmarks were related to inflammation. Single-cell RNA sequencing analyses revealed the advanced aneuploidy to be closely associated with the activation of the inflammatory-related and SASP-associated pathways. Flow cytometry-based immune profiling panel studies demonstrated that the tumor-infiltrating immune cells, such as CD8+ T, CD4+ T, PD-1+CD8+ T, and PD-1+CD4+ T cells, were significantly accumulated in the TAK-931-treated J558 mouse allografts, while the number of immune suppressive CD45+ myeloid-derived suppressor cells was significantly decreased. The single-agent treatment with TAK-931 exhibited significant antitumor efficacy and immunity in the J558 syngeneic allografts, which was confirmed by tumor re-challenging studies. Finally, TAK-931 and ICIs (anti-mPD-1, anti-mPD-L1, and anti-mCTLA-4 antibodies), in combination, were found to enhance the antiproliferative activities in the preclinical syngeneic mouse models. Conclusions: These preclinical findings suggest the therapeutic potential of TAK-931 in antitumor efficacy and immunity, which may improve clinical benefit of the currently-used immunotherapy by combination treatment. Citation Format: Tomoko Yamamori Morita, Jie Yu, Yukie Kashima, Kosuke Tanaka, Tatsunori Minamide, Chiaki Mashima, Yumi Hakozaki, Shun-ichiro Kageyama, Akito Nakamura, Eric Lightcap, Huifeng Niu, Karuppiah Kannan, Akihiro Ohashi. CDC7 inhibitor-induced replication stress generates inflamed aneuploid cells to sensitize immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5687.