Orthotopic Model Research Articles

Andrographolide sensitizes glioma to temozolomide by inhibiting DKK1 expression.

Temozolomide (TMZ) is the first-line chemotherapeutic drug for gliomas treatment. However, the clinical efficacy of TMZ in glioma patients was very limited. Therefore, it is urgently needed to discover a novel approach to increase the sensitivity of glioma cells to TMZ. Western blot, immunohistochemical staining, and qRT-PCR assays were used to explore the mechanisms underlying TMZ promoting DKK1 expression and andrographolide (AND) inhibiting DKK1 expression. HPLC was used to detect the ability of andrographolide (AND) to penetrate the blood-brain barrier. MTT assay, bioluminescence images, magnetic resonance imaging (MRI) and H&E staining were employed to measure the proliferative activity of glioma cells and the growth of intracranial tumors. TMZ can promote DKK1 expression in glioma cells and brain tumors of an orthotopic model of glioma. DKK1 could promote glioma cell proliferation and tumor growth in an orthotopic model of glioma. Mechanistically, TMZ increased EGFR expression and subsequently induced the activation of its downstream MEK-ERK and PI3K-Akt pathways, thereby promoting DKK1 expression in glioma cells. Andrographolide inhibited TMZ-induced DKK1 expression through inactivating MEK-ERK and PI3K-Akt pathways. Andrographolide can cross the blood-brain barrier, the combination of TMZ and andrographolide not only improved the anti-tumor effects of TMZ but also showed a survival benefit in an orthotopic model of glioma. Andrographolide can enhance anti-tumor activity of TMZ against glioma by inhibiting DKK1 expression.

Enhanced Tumor-Targeted Delivery of Arginine-Rich Peptides via a Positive Feedback Loop Orchestrated by Piezo1/integrin β1 Signaling Axis.

Peptide-based drugs hold great potential for cancer treatment, and their effectiveness is driven by mechanisms on how peptides target cancer cells and escape from potential lysosomal entrapment post-endocytosis. Yet, the mechanisms remain elusive, which hinder the design of peptide-based drugs. Here hendeca-arginine peptides (R11) are synthesized for targeted delivery in bladder carcinoma (BC), investigated the targeting efficiency and elucidated the mechanism of peptide-based delivery, with the aim of refining the design and efficacy of peptide-based therapeutics. It is demonstrated that the over-activated Piezo1/integrin β1 (ITGB1) signaling axis significantly facilitates tumor-targeted delivery of R11 peptides via macropinocytosis. Furthermore, R11 peptides formed hydrogen bonds with integrin β1, facilitating targeting and penetration into tumor cells. Additionally, R11 peptides protected integrin β1 from lysosome degradation, promoting its recycling from cytoplasm to membrane. Moreover, this findings establish a positive feedback loop wherein R11 peptides activate Piezo1 by increasing membrane fusion, promoting Ca2+ releasing and resulting in enhanced integrin β1-mediated endocytosis in both orthotopic models and clinical tissues, demonstrating effective tumor-targeted delivery. Eventually, the Piezo1/integrin β1 signaling axis promoted cellular uptake and transport of peptides, establishing a positive feedback loop, promoting mechanical delivery to cancer and offering possibilities for drug modification in cancer therapy.

Tumor-associated neutrophils upregulate Nectin2 expression, creating the immunosuppressive microenvironment in pancreatic ductal adenocarcinoma

BackgroundTumor-associated neutrophils (TANs) constitute an abundant component among tumor-infiltrating immune cells and have recently emerged as a critical player in pancreatic ductal adenocarcinoma (PDAC) progression. This study aimed to elucidate the pro-tumor mechanisms of TAN and identify a novel target for effective immunotherapy against PDAC.MethodsMicroarray and cytokine array analyses were performed to identify the mechanisms underlying the function of TANs. Human and mouse TANs were obtained from differentiated HL-60 cells and orthotopically transplanted PDAC tumors, respectively. The interactions of TANs with cancer and cytotoxic T-cells were evaluated through in vitro co-culture and in vivo orthotopic or subcutaneous models. Single-cell transcriptomes from patients with PDAC were analyzed to validate the cellular findings.ResultsIncreased neutrophil infiltration in the tumor microenvironment was associated with poor survival in patients with PDAC. TANs secreted abundant amounts of chemokine ligand 5 (CCL5), subsequently enhancing cancer cell migration and invasion. TANs subpopulations negatively correlated with cytotoxic CD8+ T-cell infiltration in PDAC and promoted T-cell dysfunction. TANs upregulated the membranous expression of Nectin2, which contributed to CD8+ T-cell exhaustion. Blocking Nectin2 improved CD8+ T-cell function and suppressed tumor progression in the mouse model. Single-cell analysis of human PDAC revealed two immunosuppressive TANs phenotypes: Nectin2+ TANs and OLR1+ TANs. Endoplasmic reticulum stress regulated the protumor activities in TANs.ConclusionsTANs enhance PDAC progression by secreting CCL5 and upregulating Nectin2. Targeting the immune checkpoint Nectin2 could represent a novel strategy to enhance immunotherapy efficacy in PDAC.

ZG16 enhances the maturation of dendritic cells via induction of CD40 and contributes to the antitumor immunity in pancreatic cancer.

Dendritic cells (DCs) are critical mediators of antigen priming and T-cell activation. Zymogen granule protein 16 (ZG16) is demonstrated as an anti-oncogene in T-cell mediated antitumor immunity, but its effect on DCs is largely unknown. Herein, we wonder whether ZG16 affects the activation of DCs in pancreatic cancer. Firstly, the increased ZG16 expression was observed during the maturation of DCs derived from mouse bone marrow or human peripheral blood. Then, overexpression of ZG16 or exogenous introduction of recombinant ZG16 protein induced the expression of MHC II, CD86, CD84, and CCR7 on the surface of DCs, thereby facilitating the secretion of proinflammatory mediators IL-1β, IL-6, TNF-α, and IL-12/p70, supporting the promoting effect of ZG16 on DC maturation. By establishing the subcutaneous and orthotopic mouse models of pancreatic cancer, we confirmed that intraperitoneal injection of recombinant ZG16 protein (Re-mZG16) could induce tumor regression by stimulating DC maturation and enhancing antitumor responses of CD4 + , CD8 + , PD-1 + , and Ctla4+ cells. Besides, Re-mZG16 in combination with gemcitabine showed a synergistic effect in the treatment of pancreatic cancer. Mechanistically, we demonstrated that ZG16 inhibited the ubiquitination and degradation of CD40, which depended on the lectin domain of ZG16. In conclusion, this study provided a novel insight into the role of ZG16-CD40 axis in DC-based immunotherapy for pancreatic cancer.

Enhanced radiosensitivity of pancreatic cancer achieved through inhibition of Cyclin-dependent kinase 1

Background and purposeOvercoming radioresistance is a critical challenge in pancreatic ductal adenocarcinoma (PDAC). Our study investigates the targeting of Cyclin-dependent kinase-1 (CDK1) through genetic and pharmaceutical inhibition to radiosensitize PDAC cells. Materials and MethodsMass spectrometry and phosphoproteomics were used to analyze engineered radiation-resistant PDAC cell lines (MIA PaCa-2 and PANC-1) compared to parental controls. The TCGA PDAC database was queried for clinical outcomes and patients were dichotomized based on the median CDK1 mRNA expression. We generated a microRNA-based TET-on inducible shRNA to inhibit CDK1 expression in two PDAC cell lines. We used an orthotopic model of PDAC to test the radiation sensitivity of PDAC tumors with or without doxycycline treatment. We targeted CDK1 activation with a selective CDK1 inhibitor, RO-3306, followed by in vitro experiments employing immunoblotting, immunocytochemistry, and clonogenic assays. ResultsPhosphoproteomics analysis revealed that phospho-CDK1 (Tyr15) was significantly elevated in the resistant clones. We found that high CDK1 expression was associated with worse OS in PDAC patients. Radiation exposure increased CDK1 phosphorylation. In MIA PaCa-2 and PANC-1 cells, CDK1 inhibition synergized with radiation therapy to delay tumor growth in vivo. CDK1 inhibition via. RO-3306 resulted in a significant shift of cells into the G2/M phase and disrupted DNA repair after radiation exposure. In vitro, pre-treatment with RO-3306 led to enhanced radiosensitivity of PDAC cells. ConclusionCDK1 plays a crucial role in PDAC radioresistance. Targeting CDK1 with radiotherapy holds promise for further investigation in PDAC treatment.

Inhibition of STAT3 by 2-Methoxyestradiol suppresses M2 polarization and protumoral functions of macrophages in breast cancer

BackgroundBreast cancer metastasis remains the leading cause of cancer-related deaths in women worldwide. Infiltration of tumor-associated macrophages (TAMs) in the tumor stroma is known to be correlated with reduced overall survival. The inhibitors of TAMs are sought after for reprogramming the tumor microenvironment. Signal transducer and activator of transcription 3 (STAT3) is well known to contribute in pro-tumoral properties of TAMs. 2-Methoxyestradiol (2ME2), a potent anticancer and antiangiogenic agent, has been in clinical trials for treatment of breast cancer. Here, we investigated the potential of 2ME2 in modulating the pro-tumoral effects of TAMs in breast cancer.MethodsTHP-1-derived macrophages were polarized to macrophages with or without 2ME2. The effect of 2ME2 on macrophage surface markers and anti-inflammatory genes was determined by Western blotting, flow cytometry, immunofluorescence, qRT‒PCR. The concentration of cytokines secreted by cells was monitored by ELISA. The effect of M2 macrophages on malignant properties of breast cancer cells was determined using colony formation, wound healing, transwell, and gelatin zymography assays. An orthotopic model of breast cancer was used to determine the effect of 2ME2 on macrophage polarization and metastasis in vivo.ResultsFirst, our study found that polarization of monocytes to alternatively activated M2 macrophages is associated with the reorganization of the microtubule cytoskeleton. At lower concentrations, 2ME2 treatment depolymerized microtubules and reduced the expression of CD206 and CD163, suggesting that it inhibits the polarization of macrophages to M2 phenotype. However, the M1 polarization was not significantly affected at these concentrations. Importantly, 2ME2 inhibited the expression of several anti-inflammatory cytokines and growth factors, including CCL18, TGF-β, IL-10, FNT, arginase, CXCL12, MMP9, and VEGF-A, and hindered the metastasis-promoting effects of M2 macrophages. Concurrently, 2ME2 treatment reduced the expression of CD163 in tumors and inhibited lung metastasis in the orthotopic breast cancer model. Mechanistically, 2ME2 treatment reduced the phosphorylation and nuclear translocation of STAT3, an effect which was abrogated by colivelin.ConclusionsOur study presents novel findings on mechanism of 2ME2 from the perspective of its effects on the polarization of the TAMs via the STAT3 signaling in breast cancer. Altogether, the data supports further clinical investigation of 2ME2 and its derivatives as therapeutic agents to modulate the tumor microenvironment and immune response in breast carcinoma.

Performance of PSMA-targeted radiotheranostics in an experimental model of renal cell carcinoma.

Renal cell carcinoma (RCC) represents cancer originating from the renal epithelium and accounts for > 90% of cancers in the kidney. Prostate-specific membrane antigen (PSMA) is overexpressed in tumor-associated neovascular endothelial cells of many solid tumors, including metastatic RCC. Although studied in several small clinical studies, PSMA-based imaging and therapy have not been pursued rigorously in preclinical RCC. This study aimed to evaluate the preclinical performance of PSMA-based radiotheranostic agents in a relevant murine model. A PSMA-overexpressing murine cell line, PSMA+ RENCA, was developed by lentiviral transduction. PSMA-based theranostic agents, 68Ga-L1/177Lu-L1/225Ac-L1, were synthesized in high radiochemical yield and purity following our reported methods. Immunocompetent BALB/c mice were used for flank and orthotopic tumor inoculation. 68Ga-L1 was evaluated in small animal PET/CT imaging in flank and PET/MR imaging in orthotopic models. Cell viability studies were conducted for 177Lu-L1 and 225Ac-L1. Proof-of-concept treatment studies were performed using 225Ac-L1 (0, 37 kBq, 2 kBq × 37 kBq, 1 week apart) using PSMA+ RENCA in the flank model. Cellular uptake of 68Ga-L1, 177Lu-L1, and 225Ac-L1 confirmed the specificity of the agents to PSMA+ RENCA cells rather than to RENCA (wt) cells, which are low in PSMA expression. The uptake in PSMA+ RENCA cells at 1 h for 68Ga-L1 (49.0% incubated dose [ID] ± 3.6%ID/million cells), 177Lu-L1 (22.1%ID ± 0.5%ID)/million cells), and 225Ac-L1 (4.1% ± 0.2% ID)/million cells), respectively, were higher than the RENCA (wt) cells (~ 1%ID-2%ID/million cells). PET/CT images displayed > 7-fold higher accumulation of 68Ga-L1 in PSMA+ RENCA compared to RENCA (wt) in flank implantation at 1 h. A twofold higher accumulation of 68Ga-L1 was observed in orthotopic tumors than in normal kidneys during 1-3 h postinjection. High lung uptake was observed with 68Ga-L1 PET/MR imaging 3 weeks after orthotopic implantation of PSMA+ RENCA due to spontaneous lung metastases. The imaging data were further confirmed by immunohistochemical characterization. 225Ac-L1 (0-37 kBq) displayed a dose-dependent reduction of cell proliferation in the PSMA+ RENCA cells after 48 h incubation; ~ 40% reduction in the cells with treated 37 kBq compared to vehicle (p < 0.001); however, no effect was observed with 177Lu-L1 (0-3700 kBq) up to 144 h postinoculation, suggesting lower efficacy of β-particle-emitting radiations in cellular studies compared to α-particle-emitting 225Ac-L1. Animals treated with 225Ac-L1 at 1 week posttumor inoculation in flank models displayed significant tumor growth delay (p < 0.03) and longer median survival of 21 days and 24 days for the treatment groups 37 kBq and 2 kBq × 37 kBq, respectively, compared to the vehicle group (12 days). The results suggest that a theranostic strategy targeting PSMA, employing PET and α-emitting radiopharmaceuticals, enabled tumor growth control and enhanced survival in a relevant immunocompetent murine model of RCC. These studies provide the rationale for clinical studies of PSMA-targeted theranostic agents in patients with RCC.

GOLM1 dictates acquired Lenvatinib resistance by a GOLM1-CSN5 positive feedback loop upon EGFR signaling activation in hepatocellular carcinoma.

Lenvatinib is a multiple receptor tyrosine kinases inhibitor (TKI) authorized for first-line treatment of hepatocellular carcinoma (HCC). However, Lenvatinib resistance is common in HCC clinical treatment, highlighting the urgent need to understand mechanisms of resistance. Here, we identified Golgi membrane protein 1 (GOLM1), a type II transmembrane protein originally located in the Golgi apparatus, as a novel regulator of Lenvatinib resistance. We found GOLM1 was overexpressed in Lenvatinib resistant human HCC cell lines, blood and HCC samples. Additionally, GOLM1 overexpression contributes to Lenvatinib resistance and HCC progression in vitro and in vivo. Mechanistically, GOLM1 upregulates CSN5 expression through EGFR-STAT3 pathway. Reversely, CSN5 deubiquitinates and stabilizes GOLM1 protein by inhibiting ubiquitin-proteasome pathway of GOLM1. Furthermore, clinical specimens of HCC showed a positive correlation between the activation of the GOLM1-EGFR-STAT3-CSN5 axis. Finally, GOLM1 knockdown was found to act in synergy with Lenvatinib in subcutaneous and orthotopic mouse model. Overall, these findings identify a mechanism of resistance to Lenvatinib treatment for HCC, highlight an effective predictive biomarker of Lenvatinib response in HCC and show that targeting GOLM1 may improve the clinical benefit of Lenvatinib.

Three-dimensional analysis of mitochondria in a patient-derived xenograft model of triple negative breast cancer reveals mitochondrial network remodeling following chemotherapy treatments.

Mitochondria are hubs of metabolism and signaling and play an important role in tumorigenesis, therapeutic resistance, and metastasis in many cancer types. Various laboratory models of cancer demonstrate the extraordinary dynamics of mitochondrial structure, but little is known about the role of mitochondrial structure in resistance to anticancer therapy. We previously demonstrated the importance of mitochondrial structure and oxidative phosphorylation in the survival of chemotherapy-refractory triple negative breast cancer (TNBC) cells. As TNBC is a highly aggressive breast cancer subtype with few targeted therapy options, conventional chemotherapies remain the backbone of early TNBC treatment. Unfortunately, approximately 45% of TNBC patients retain substantial residual tumor burden following chemotherapy, associated with abysmal prognoses. Using an orthotopic patient-derived xenograft mouse model of human TNBC, we compared mitochondrial structures between treatment-naïve tumors and residual tumors after conventional chemotherapeutics were administered singly or in combination. We reconstructed 1,750 mitochondria in three dimensions from serial block-face scanning electron micrographs, providing unprecedented insights into the complexity and intra-tumoral heterogeneity of mitochondria in TNBC. Following exposure to carboplatin or docetaxel given individually, residual tumor mitochondria exhibited significant increases in mitochondrial complexity index, area, volume, perimeter, width, and length relative to treatment-naïve tumor mitochondria. In contrast, residual tumors exposed to those chemotherapies given in combination exhibited diminished mitochondrial structure changes. Further, we document extensive intra-tumoral heterogeneity of mitochondrial structure, especially prior to chemotherapeutic exposure. These results highlight the potential for structure-based monitoring of chemotherapeutic responses and reveal potential molecular mechanisms that underlie chemotherapeutic resistance in TNBC.

FHL2 expression by cancer-associated fibroblasts promotes metastasis and angiogenesis in lung adenocarcinoma.

Cancer-associated fibroblasts (CAFs) contribute to the progression of lung cancer. Four and a half LIM domain protein-2 (FHL2) is a component of focal adhesion structures. We analyzed the function of FHL2 expressed by CAFs in lung adenocarcinoma. Expression of FHL2 in fibroblast subtypes was investigated using database of single-cell RNA-sequencing of lung cancer tissue. The role of FHL2 in the proliferation and migration of CAFs was assessed. The effects of FHL2 knockout on the migration and invasion of human lung adenocarcinoma cells and tube formation of endothelial cells induced by CAF-conditioned medium (CM) were evaluated. The effect of FHL2 knockout in CAFs on metastasis was determined using a murine orthotopic lung cancer model. The prognostic significance of stromal FHL2 was assessed by immunohistochemistry in human adenocarcinoma specimens. FHL2 is highly expressed in myofibroblasts in cancer tissue. TGF-β1 upregulated FHL2 expression in CAFs and FHL2 knockdown attenuated CAF proliferation. FHL2 knockout reduced CAF induced migration of A110L and H23 human lung adenocarcinoma cell lines, and the induction of tube formation of endothelial cells. FHL2 knockout reduced CAF-induced metastasis of lung adenocarcinomas in an orthotopic model in vivo. The concentration of Osteopontin (OPN) in CM from CAF was downregulated by FHL2 knockout. siRNA silencing and antibody blocking of OPN reduced the pro-migratory effect of CM from CAF on lung cancer cells. In resected lung adenocarcinoma specimens, positive stromal FHL2 expression was significantly associated with higher microvascular density and worse prognosis. In conclusion, FHL2 expression by CAFs enhances the progression of lung adenocarcinoma by promoting angiogenesis and metastasis.

HKDC1 promotes liver cancer stemness under hypoxia through stabilizing β-catenin.

Hexokinases (HKs), a group of enzymes catalyzing the first step of glycolysis, have been shown to play important roles in liver metabolism and tumorigenesis. Our recent studies identified hexokinase domain containing 1 (HKDC1) as a top candidate associated with liver cancer metastasis. We aimed to compare its cell-type specificity with other HKs upregulated in liver cancer and investigate the molecular mechanisms underlying its involvement in liver cancer metastasis. We found that, compared to HK1 and HK2, the other 2 commonly upregulated HKs in liver cancer, HKDC1 was most strongly associated with the metastasis potential of tumors and organoids derived from 2 liver cancer mouse models we previously established. RNA in situ hybridization and single-cell RNA-seq analysis revealed that HKDC1 was specifically upregulated in malignant cells in HCC and cholangiocarcinoma patient tumors, whereas HK1 and HK2 were widespread across various tumor microenvironment lineages. An unbiased metabolomic profiling demonstrated that HKDC1 overexpression in HCC cells led to metabolic alterations distinct from those from HK1 and HK2 overexpression, with HKDC1 particularly impacting the tricarboxylic acid cycle. HKDC1 was prometastatic in HCC orthotopic and tail vein injection mouse models. Molecularly, HKDC1 was induced by hypoxia and bound to glycogen synthase kinase 3β to stabilize β-catenin, leading to enhanced stemness of HCC cells. Overall, our findings underscore HKDC1 as a prometastatic HK specifically expressed in the malignant compartment of primary liver tumors, thereby providing a mechanistic basis for targeting this enzyme in advanced liver cancer.

Targeting stress induction of GRP78 by cardiac glycoside oleandrin dually suppresses cancer and COVID-19

BackgroundDespite recent therapeutic advances, combating cancer resistance remains a formidable challenge. The 78-kilodalton glucose-regulated protein (GRP78), a key stress-inducible endoplasmic reticulum (ER) chaperone, plays a crucial role in both cancer cell survival and stress adaptation. GRP78 is also upregulated during SARS-CoV-2 infection and acts as a critical host factor. Recently, we discovered cardiac glycosides (CGs) as novel suppressors of GRP78 stress induction through a high-throughput screen of clinically relevant compound libraries. This study aims to test the possibility that agents capable of blocking stress induction of GRP78 could dually suppress cancer and COVID-19.ResultsHere we report that oleandrin (OLN), is the most potent among the CGs in inhibiting acute stress induction of total GRP78, which also results in reduced cell surface and nuclear forms of GRP78 in stressed cells. The inhibition of stress induction of GRP78 is at the post-transcriptional level, independent of protein degradation and autophagy and may involve translational control as OLN blocks stress-induced loading of ribosomes onto GRP78 mRNAs. Moreover, the human Na+/K+-ATPase α3 isoform is critical for OLN suppression of GRP78 stress induction. OLN, in nanomolar range, enhances apoptosis, sensitizes colorectal cancer cells to chemotherapeutic agents, and reduces the viability of patient-derived colon cancer organoids. Likewise, OLN, suppresses GRP78 expression and impedes tumor growth in an orthotopic breast cancer xenograft model. Furthermore, OLN blocks infection by SARS-CoV-2 and its variants and enhances existing anti-viral therapies. Notably, GRP78 overexpression mitigates OLN-mediated cancer cell apoptotic onset and suppression of virus release.ConclusionOur findings validate GRP78 as a target of OLN anti-cancer and anti-viral activities. These proof-of-principle studies support further investigation of OLN as a readily accessible compound to dually combat cancer and COVID-19.

Abstract B053: Tumor-associated macrophages diminishes IL1RAP-targeting CAR-T cell therapy efficacy in Ewing sarcoma

Abstract Ewing sarcoma (EwS) is a pediatric malignancy of soft tissue and bone with a poor prognosis, especially in its metastatic stages. We have previously identified IL1RAP as a promising target for immunotherapies like chimeric antigen receptor (CAR) T cell therapy against EwS. However, CAR-T cells, despite their success in hematological cancers, often show limited efficacy against solid tumors. In orthotopic xenograft models of Ewing sarcoma (EwS) we have observed that CD11b+ CD206+ macrophages, known as tumor-associated macrophages (TAMs), surround the primary tumor. Treatment with IL1RAP-targeting CAR-T cells specific to EwS results in T cells localizing with these macrophages and failing to infiltrate the primary tumor. This study aims to characterize the impact of TAMs on CAR-T cell efficacy in EwS. We hypothesized that TAMs, polarized by Ewing sarcoma cells, suppress IL1RAP CAR-T cell activity. To model these interactions, we co-cultured EwS cells, macrophages, and CAR-T cells, performing luciferin-based in vitro cytotoxicity assays. THP-1 monocytes were differentiated into macrophages and polarized into M1 or M2 states using cytokines (LPS + IFN-y, IL-4 + IL-13), or differentiated into TAMs by co-culture with EwS cell lines. In vitro cytotoxicity assays were conducted using luciferase-expressing EwS target cells, polarized THP-1 macrophages, and IL1RAP CAR-T effector cells, with luminescence indicating EwS cell survival. We found that M1 macrophages did not affect CAR-T cell targeting of EwS cells, whereas M2 macrophages and TAMs significantly decreased specific lysis (p &amp;lt; 0.001). Conditioned media from polarized macrophages had no effect on CAR-T cell performance against EwS, suggesting that physical proximity or direct contact with TAMs is necessary to suppress CAR-T cell function. Western blot analysis of polarized THP-1 macrophages revealed that M2-polarized macrophages upregulated IL1RAP expression compared to M1 macrophages. Ongoing experiments aim to determine whether elevated IL1RAP expression in M2 macrophages redirects IL1RAP CAR-T cells away from EwS cells or if M2 macrophages mediate general suppression of CAR-T cell activity. Additionally, we are investigating whether THP-1-derived TAMs and primary macrophages also upregulate IL1RAP expression when polarized and how this affects syngeneic CAR-T cell performance. Our findings indicate that TAMs can influence the efficacy of IL1RAP-targeting CAR-T cells against EwS. Further investigation into the specific mechanisms responsible for this suppressive effect could enhance CAR-T therapy for Ewing sarcoma and potentially other solid tumors. Citation Format: Yue Zhou (Joe) Huang, Melanie Rouleu, Poul Sorensen. Tumor-associated macrophages diminishes IL1RAP-targeting CAR-T cell therapy efficacy in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B053.

A Novel Liver Cancer-Selective Histone Deacetylase Inhibitor Is Effective against Hepatocellular Carcinoma and Induces Durable Responses with Immunotherapy.

Hepatocellular carcinoma (HCC) progression is facilitated by gene-silencing chromatin histone hypoacetylation due to histone deacetylase (HDAC) activation. However, inhibiting HDACs-an effective treatment for lymphomas-has shown limited success in solid tumors. We report the discovery of a class of HDAC inhibitors (HDACi) that demonstrates exquisite selective cytotoxicity against human HCC cells. The lead compound STR-V-53 (3) showed a favorable safety profile in mice and robustly suppressed tumor growth in orthotopic xenograft models of HCC. When combined with the anti-HCC drug sorafenib, STR-V-53, showed greater in vivo efficacy. Moreover, STR-V-53 combined with anti-PD1 therapy increased the CD8+ to regulatory T-cell (Treg) ratio and survival in an orthotopic HCC model in immunocompetent mice. This combination therapy resulted in durable responses in 40% of the mice. Transcriptomic analysis revealed that STR-V-53 primed HCC cells to immunotherapy through HDAC inhibition, impaired glucose-regulated transcription, impaired DNA synthesis, upregulated apoptosis, and stimulated the immune response pathway. Collectively, our data demonstrate that the novel HDACi STR-V-53 is an effective anti-HCC agent that can induce profound responses when combined with standard immunotherapy.

The dual role of POSTN in maintaining glioblastoma stem cells and the immunosuppressive phenotype of microglia in glioblastoma

BackgroundGlioblastoma (GBM) is an immunosuppressive, universally lethal cancer driven by glioblastoma stem cells (GSCs). The interplay between GSCs and immunosuppressive microglia plays crucial roles in promoting the malignant growth of GBM; however, the molecular mechanisms underlying this crosstalk are unclear. This study aimed to investigate the role of POSTN in maintaining GSCs and the immunosuppressive phenotype of microglia.MethodsThe expression of POSTN in GBM was identified via immunohistochemistry, quantitative real-time PCR, and immunoblotting. Tumorsphere formation assay, Cell Counting Kit-8 assay and immunofluorescence were used to determine the key role of POSTN in GSC maintenance. ChIP-seq and ChIP-PCR were conducted to confirm the binding sequences of β-catenin in the promoter region of FOSL1. Transwell migration assays, developmental and functional analyses of CD4+ T cells, CFSE staining and analysis, enzyme-linked immunosorbent assays and apoptosis detection tests were used to determine the key role of POSTN in maintaining the immunosuppressive phenotype of microglia and thereby promoting the immunosuppressive tumor microenvironment. Furthermore, the effects of POSTN on GSC maintenance and the immunosuppressive phenotype of microglia were investigated in a patient-derived xenograft model and orthotopic glioma mouse model, respectively.ResultsOur findings revealed that POSTN secreted from GSCs promotes GSC self-renewal and tumor growth via activation of the αVβ3/PI3K/AKT/β-catenin/FOSL1 pathway. In addition to its intrinsic effects on GSCs, POSTN can recruit microglia and upregulate CD70 expression in microglia through the αVβ3/PI3K/AKT/NFκB pathway, which in turn promotes Treg development and functionality and supports the formation of an immunosuppressive tumor microenvironment. In both in vitro models and orthotopic mouse models of GBM, POSTN depletion disrupted GSC maintenance, decreased the recruitment of immunosuppressive microglia and suppressed GBM growth.ConclusionOur findings reveal that POSTN plays critical roles in maintaining GSCs and the immunosuppressive phenotype of microglia and provide a new therapeutic target for treating GBM.

An Oxidative Stress Nano-Amplifier for Improved Tumor Elimination and Combined Immunotherapy.

Amplifying oxidative stress to disrupt intracellular redox homeostasis can accelerate tumor cell death. In this work, an oxidative stress amplifier (PP@T) is prepared for enhanced tumor oxidation therapy to reduce tumor growth and metastases. The nano-amplifier has been successfully constructed by embedding MTH1 inhibitor (TH588) in the PDA-coated porphyrin metal-organic framework PCN-224. The controllable-released TH588 is demonstrated from pores can hinder MTH1-mediated damage-repairing process by preventing the hydrolysis of 8-oxo-dG, thereby amplifying oxidative stress and exacerbating the oxidative DNA damage induced by the sonodynamic therapy of PP@T under ultrasound irradiation. Furthermore, PP@T can effectively induce immunogenic cell death to trigger systemic anti-tumor immune response. When administered in combination with immune checkpoint blockade, PP@T not only impedes the progression of the primary tumor but also achieves obvious antimetastasis in breast cancer murine models, including orthotopic and artificial whole-body metastasis models. Furthermore, the nanoplatform also provides photoacoustic imaging for in vivo treatment guidance. In conclusion, by amplifying oxidative stress and reactive oxygen species sensitized immunotherapy, this image-guided nanosystem shows potential for highly specific, effective combined therapy against tumor cells with negligible side-effects to normal cells which will provide a new insight for precise tumor treatment.

385. TRANSLATING MOLECULAR-TARGETING FLUORESCENT PROBES IN OESOPHAGEAL CANCER PRE-CLINICAL MODELS

Abstract Background Intra-operative molecular imaging (IMI) is an emerging field that utilises tumour-targeting fluorescent probes to improve oncological outcomes. A novel class of probe known as quenched activity-based probes (qABP), developed to measure enzyme activity in-vitro, has been repurposed to target tumour-expressed proteases known as cathepsins. Unlike other tumour-targeting probes, fluorescence is inhibited up until the qABP covalently bonds to cathepsins, releasing an inhibitory quencher which results in fluorescence. This may improve the contrast between normal tissue and tumour. Our laboratory group sought to obtain pre-clinical evidence to eventually translate qABP’s into the operating room for oesophageal, junctional and gastric cancer. Methods Cathepsin activity was screened in cell lines from the normal oesophagus, Barrett’s metaplasia, oesophageal adenocarcinoma (OAC), squamous cell carcinoma (SCC) and gastric adenocarcinoma (GAC) with two qABP’s known as BMV109 and VGT309. We additionally screened patient biopsies from oesophageal, junctional and gastric cancers. Cell line and tissue samples were analysed using in-gel fluorescence (Amersham Typhoon®) and Western blot. In-vivo, cell line xenografts were established in NSG mice using subcutaneous and orthotopic models. Tumour-bearing mice were then injected with VGT-309 and then imaged at specific timepoints using the IVIS® spectrum imager. Results Cathepsin activity was found in all oesophago-gastric cell lines and selectivity proven with a cathepsin inhibitor. Matched biopsies were collected from patients prior before and after neoadjuvant chemo-radiotherapy. Baseline tumour biopsies exhibited significantly higher cathepsin activity than normal biopsies, with these differences preserved in biopsies collected after chemo-radiotherapy (p &amp;lt;0.001). In-vivo, OAC and GAC xenografts were identified in NSG mice as early as 12 hours post injection of VGT-309, exhibiting a 2.5-fold increase in fluorescence compared to normal background stomach. Bio-distribution analysis demonstrated that VGT-309 accumulated in liver and kidney, but less so in the murine oesophagus and stomach. Conclusion Translating qABP’s into the operating room has the potential to detect early cancers in Barrett’s metaplasia, reduce positive margin rates and identify lymph node metastasis. Our research group continues to investigate qABP’s, including determining their sensitivity to detect lymph node metastasis with the aim of translating this technology into a phase 1 clinical trial.

Low-dose orthotopic cancer implantation permits measurement of longitudinal functional changes associated with cachexia.

Progressive functional decline is a key element of cancer-associated cachexia. Major barriers to translating preclinical therapies into the clinic include lack of cancer models that accurately mimic functional decline, which develops over time, and use of nonspecific measures, like grip strength, as surrogates for physical function. In this study, we aimed to extend the survival and longevity of a cancer model, to investigate cachexia-related function at the basic science level. Survival extension studies were performed by testing multiple cell lines, dilutions, and vehicle-types in orthotopic implantation of K-rasLSL.G12D/+; Trp53R172H/+; Pdx-1-Cre (KPC)-derived cells. One hundred twenty-eight animals in this new model were assessed for cachexia syndrome phenotype using a battery of anatomical, biochemical, and behavioral techniques. We extended the survival of the KPC orthotopic model to 8-9 wk postimplantation using a relatively low 100-cell dose of DT10022 KPC cells (P < 0.001). In this low-dose orthotopic (LO) model, progressive muscle wasting was detected in parallel to systemic inflammation; skeletal muscle atrophy at the fiber level was detected as early as 3 wk postimplantation compared with controls (P < 0.001). Gait speed in LO animals declined as early as 2 wk postimplantation, whereas grip strength change was a late event. Principal component and regression analyses revealed distinct cachectic and noncachectic animal populations, which we leveraged to show that the gait speed decline was specific to cachexia (P < 0.01), whereas grip strength decline was not (P = 0.19). Gait speed represents an accurate surrogate for cachexia-related physical function as opposed to grip strength.NEW & NOTEWORTHY Previous studies of cancer-induced cachexia have been confounded by the relatively rapid death of animal subjects. Using a lower dose of cancer cells in combination with a battery of behavioral, structural, histological, and biochemical techniques, we show that gait speed is actually the best indicator of functional decline due to cachexia. Future studies are required to define the underlying physiological basis of these findings.

20(S)-Ginsenoside Rh2 overcomes gemcitabine resistance in pancreatic cancer by inhibiting LAMC2-Modulated ABC transporters

IntroductionGemcitabine (GEM) is the first-line drug for pancreatic ductal adenocarcinoma (PDAC), but drug resistance severely restricts its chemotherapeutic efficacy. Laminin subunit γ2 (LAMC2) plays a crucial role in extracellular matrix formation in the development of GEM-resistance. However, the biological function of LAMC2 in GEM resistance and its molecular mechanisms are still unclear. 20(S)-Ginsenoside Rh2 (Rh2), one of the principal active components isolated from Ginseng Radix et Rhizoma, possesses strong anti-tumor effects. However, the effects of Rh2 on overcoming GEM resistance and its action mechanisms remain to be elucidated. ObjectivesThis study aimed to determine the efficacy of Rh2 on overcoming GEM resistance and to explore its underlying molecular mechanisms. MethodsClinical study, Western blotting, publicly available databasesand bioinformatic analyses were performed to investigate the protein expression of LAMC2 in the GEM-resistant PDAC patients and the acquired GEM-resistant PDAC cells. Then, the effects of Rh2 on overcoming the GEM resistance in PDAC were evaluated both in vitro and in vivo. Stable silencing or overexpression of LAMC2 in the GEM-resistant PDAC cells were established for validating the role of LAMC2 on Rh2 overcoming the GEM resistance in PDAC. ResultsThe protein expression of LAMC2 was markedly increased in the GEM-resistant PDAC patient biopsies compared to the sensitive cases. The protein expression of LAMC2 was significantly higher in the acquired GEM-resistant PDAC cells than that in their parental cells. Rh2 enhanced the chemosensitivity of GEM in the GEM-resistant PDAC cells, and inhibited the tumor growth of Miapaca-2-GR cell-bearing mice and Krastm4TyjTrp53tm1BrnTg (Pdx1-cre/Esr1*) #Dam/J (KPC) mice. Rh2 effectively reversed the GEM resistance in Miapaca-2-GR and Capan-2-GR cells by inhibiting LAMC2 expression through regulating the ubiquitin–proteasome pathway. Knockdown of LAMC2 enhanced the chemosensitivity of GEM and the effects of Rh2 on overcoming the GEM resistance in PDAC cells and the orthotopic PDAC mouse model. Conversely, LAMC2 overexpression aggravated the chemoresistance of GEM and abolished the effects of Rh2 on overcoming GEM resistance via modulating ATP-binding cassette (ABC) transporters leading to the active GEM efflux. ConclusionsLAMC2 plays an important role in the GEM resistance in PDAC, and Rh2 is a potential adjuvant for overcoming the chemoresistance of GEM in PDAC.

CD94+ natural killer cells potentiate pulmonary ischaemia-reperfusion injury.

Pulmonary ischaemia-reperfusion injury (IRI) is a major contributor to poor lung transplant outcomes. We recently demonstrated a central role of airway-centred natural killer (NK) cells in mediating IRI; however, there are no existing effective therapies for directly targeting NK cells in humans. We hypothesised that a depleting anti-CD94 monoclonal antibody (mAb) would provide therapeutic benefit in mouse and human models of IRI based on high levels of KLRD1 (CD94) transcripts in bronchoalveolar lavage samples from lung transplant patients. We found that CD94 is highly expressed on mouse and human NK cells, with increased expression during IRI. Anti-mouse and anti-human mAbs against CD94 showed effective NK cell depletion in mouse and human models and blunted lung damage and airway epithelial killing, respectively. In two different allogeneic orthotopic lung transplant mouse models, anti-CD94 treatment during induction reduced early lung injury and chronic inflammation relative to control therapies. Anti-CD94 did not increase donor antigen-presenting cells that could alter long-term graft acceptance. Lung transplant induction regimens incorporating anti-CD94 treatment may safely improve early clinical outcomes.