Survival Of Tumor-bearing Mice Research Articles

Antineoplastic Activity of Sodium Caseinate in a Cytarabine-Resistant Mouse Acute Myeloid Leukemia Cell Line

Background: Acute myeloid leukemia (AML) is a hematological neoplasm of rapid and progressive onset, and is the most common form of leukemia in adults. Chemoresistance to conventional treatments such as cytarabine (Ara-C) and daunorubicin is a main cause of relapse, recurrence, metastasis, and high mortality in AML patients. It is known that sodium caseinate (SC), a salt derived from casein, a milk protein, inhibits growth and induces apoptosis in acute myeloid leukemia cells but not in normal hematopoietic cells. However, it is unknown whether SC retains its antileukemic effect in cytarabine-resistant AML cell lines. Objective: To evaluate the antineoplastic effect of SC in cytarabine-resistant leukemia models. Methods: The SC inhibits the growth and induces apoptosis in parental WEHI-3 AML cells. Here, we generated two cytarabine-resistant sublines, WEHI-CR25 and WEHI-CR50, which exhibit 6- and 16-fold increased resistance to cytarabine, respectively, compared to the parental WEHI-3 cells. Thus, these sublines mimic a chemoresistant model. Results: We demonstrate that WEHI-CR25 and WEHI-CR50 cells retain sensitivity to SC, similar to parental WEHI-3 cells. This sensitivity results in inhibited cell proliferation, induced apoptosis, and increased expression of ENT1 and dCK, molecules involved in the entry and metabolism of Ara-C, while decreasing MDR1 expression. Additionally, we observed that SC prolonged the survival of WEHI-CR50 tumor-bearing mice, despite their resistance to Ara-C. Conclusion: This is the first evidence that SC, a milk protein, may inhibit proliferation and induce apoptosis in cytarabine-resistant cells.

Divergent effects of acute and chronic PPT1 inhibition in melanoma

ABSTRACT Macroautophagy/autophagy-lysosome function promotes growth and survival of cancer cells, making them attractive targets for cancer therapy. One intriguing lysosomal target is PPT1 (palmitoyl-protein thioesterase 1). PPT1 inhibitors derived from chloroquine block autophagy, have significant antitumor activity in preclinical models and are being developed for clinical trials. However, the role of PPT1 in tumorigenesis remains poorly understood. Here we report that in melanoma cells, acute siRNA or pharmacological PPT1 inhibition led to increased ferroptosis sensitivity and significant loss of viability, whereas chronic PPT1 knockout using CRISPR-Cas9 produced blunted ferroptosis that led to sustained viability and growth. Each mode of PPT1 inhibition produced lysosome-autophagy inhibition but distinct proteomic changes, demonstrating the complexity of cellular adaptation mechanisms. To determine whether total genetic loss of Ppt1 would affect tumorigenesis in vivo, we developed a Ppt1 conditional knockout mouse model. We then crossed it into the BrafCA, PtenloxP, Tyr:CreERT2 melanoma mouse model to investigate the impact of Ppt1 loss on tumorigenesis. Loss of Ppt1 had no impact on melanoma histology, time to tumor initiation, or survival of tumor-bearing mice. These results suggest that chemical PPT1 inhibitors produce different adaptations than genetic PPT1 inhibition, and additional studies are warranted to fully understand the mechanism of chloroquine derivatives that target PPT1 in cancer.Abbreviations: 4-HT: 4-hydroxytamoxifen; BRAF: B-Raf proto-oncogene, serine/threonine kinase; cKO: conditional knockout; CRISPR-Cas9: clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9; DC661: A specific PPT1 inhibitor; DMSO: dimethyl sulfoxide; Dox; doxycycline hyclate; Easi-CRISPR: efficient additions with ssDNA inserts-CRISPR; GNS561/ezurpimtrostat: A PPT1 inhibitor; Hug: human guide; iCas: inducible CRISPR-Cas9; KO: knockout; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; LDLR: low density lipoprotein receptor; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; NT: non-target; PTEN: phosphatase and tensin homolog; PPT1: palmitoyl-protein thioesterase 1; RSL3: RAS-selective lethal small molecule 3; SCRIB/SCRB1: scribble planar cell polarity protein; Tyr:CreERT2: tyrosinase-driven Cre recombinase fused with the tamoxifen-inducible mutant ligand binding domain of the human estrogen receptor; UGCG: UDP-glucose ceramide glucosyltransferase; WT: wild-type.

Regression of renal cell carcinoma by T cell receptor-engineered T cells targeting a human endogenous retrovirus

BackgroundWe discovered a novel human endogenous retrovirus (CT-RCC HERV-E) that was selectively expressed in most clear cell renal cell carcinomas (ccRCC) and served as a source of antigens for T...

Stepwise-targeting and hypoxia-responsive liposome AMVY@NPs carrying siYAP and verteporfin for glioblastoma therapy

BackgroundThe Hippo pathway is a conserved tumour suppressor signalling pathway, and its dysregulation is often associated with abnormal cell growth and tumorigenesis. We previously revealed that the transcriptional coactivator Yes-associated protein (YAP), the key effector of the Hippo pathway, is a molecular target for glioblastoma (GBM), the most common malignant brain tumour. Inhibiting YAP with small interfering RNA (siYAP) or the specific inhibitor verteporfin (VP) can diminish GBM growth to a certain degree.ResultsIn this study, to enhance the anti-GBM effect of siYAP and VP, we designed stepwise-targeting and hypoxia-responsive liposomes (AMVY@NPs), which encapsulate hypoxia-responsive polymetronidazole-coated VP and DOTAP adsorbed siYAP, with angiopep-2 (A2) modification on the surface. AMVY@NPs exhibited excellent blood‒brain barrier crossing, GBM targeting, and hypoxia-responsive and efficient siYAP and VP release properties. By inhibiting the expression and function of YAP, AMVY@NPs synergistically inhibited both the growth and stemness of GBM in vitro. Moreover, AMVY@NPs strongly inhibited the growth of orthotopic U87 xenografts and improved the survival of tumour-bearing mice without adverse effects.ConclusionSpecific targeting of YAP with stepwise-targeting and hypoxia-responsive liposome AMVY@NPs carrying siYAP and VP efficiently inhibited GBM progression. This study provides a valuable drug delivery platform and creative insights for molecular targeted treatment of GBM in the future.

Reprogramming tumor immune microenvironment by milbemycin oxime results in pancreatic tumor growth suppression and enhanced anti-PD-1 efficacy

Pancreatic ductal adenocarcinoma (PDAC) has a survival rate of 12% and multiple clinical trials testing anti-PD1 therapies against PDAC have failed suggesting a need of novel therapeutic strategy. In this study, we evaluated the potential of milbemycin oxime (MBO), an antiparasitic compound, as an immunomodulatory agent in PDAC. Our results show that MBO inhibited growth of multiple PDAC cell lines by inducing apoptosis. In vivo studies showed that oral administration of 5 mg/kg MBO inhibited PDAC tumor growth in both subcutaneous and orthotopic models by 49 and 56%, respectively. Additionally, MBO treatment significantly increased the survival of tumor-bearing mice by 27 days as compared to control group. Interestingly, tumors from MBO-treated mice had increased infiltration of CD8+ T cells. Notably, depletion of CD8+ T cells significantly reduced the anti-tumor efficacy of MBO in mice. Further, MBO significantly augmented the efficacy of anti-PD1 therapy, and the combination treatment resulted in a greater proportion of active cytotoxic T cells within the TME. MBO was safe and well tolerated in all our preclinical toxicological studies. Overall, our study provides a new direction for the use of MBO against PDAC and highlights the potential of repurposing MBO for enhancing anti-PD1 immunotherapy.

Spore Oil enhances the effect of cyclophosphamide inhibiting programmed death-1 and prolongs the survival of H22 tumor-bearing mice.

To investigate the effect of Ganoderma Lucidum Spore Oil (GLSO) on the tumor growth and survival of H22 tumor-bearing mice treated with cyclophosphamide (CTX), and explore the underlying mechanism. Allograft H22 hepatocellular carcinoma mouse model was applied to investigate the effect of GLSO on the tumor growth and survival of animals, and Kaplan-Meier survival analysis was used to analyze the life span. Plasma biochemical examination was used to determine the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (UREA) and creatinine (CRE). Western blot analysis was performed to detect Programmed Death-1 (PD-1), Programmed Death Ligand 1 (PD-L1), Janus Kinase 2 (JAK2), phosphorylated Signal Transducer and Activator of Transcription 3 (p-STAT3), and Signal Transducer and Activator of Transcription 3 (STAT3) expression. GLSO increased the anti-tumor effect of CTX and prolonged the survival of H22 tumor-bearing mice treated with CTX. Meanwhile, GLSO increased the thymus index and showed no obvious toxicity to liver functions of animals. GLSO also decreased the level of UREA in H22 tumor-bearing mice treated with CTX. Furthermore, GLSO could inhibit the expression of PD-1 in spleen, which was independent of JAK2 expression and STAT3 phosphorylation. However, GLSO did not affect the expression of PD-L1, JAK2, and p-STAT3 in tumor tissue. GLSO could strengthen the anti-tumor effect of CTX and prolong the life span of H22 tumor-bearing mice, while the underlying mechanism might be relevant to the amelioration effect of thymus function and inhibition of PD-1 expression in spleen. Furthermore, these findings implied the promising role of GLSO in combination with CTX to extend the survival of patients in clinical chemotherapy of hepatocellular carcinoma.

Intranuclear Irradiation Inhibits Solid Tumor Growth by Upregulating Caspase8 and Activating Apoptosis.

Radioimmunotherapy (RIT) is a novel and promising cancer treatment method, with ongoing research focusing on RIT antibody selection, radionuclides, treatment options, and benefited patient groups. As we dive into the mechanisms of tumor biology, a deeper exploration of how RIT affects tumor tissue is needed to provide new ways to improve clinical treatment outcome and patient prognosis. We labeled the anti-PD-L1 monoclonal antibody atezolizumab with iodine-131 (131I), separated and purified the labeled mAb with Sephadex G-25 medium gel filtration resin, and tested product stability. We detected the in vivo activity of 131I-PD-L1 mAb by analyzing its in vivo biodistribution and performing SPECT imaging and then set different treatment groups to study the effect of 131I-atezolizumab on the survival of tumor-bearing mice. Western blot, real-time quantitative PCR, and immunohistochemistry were used to detect the expression level of Caspase8 and Nlrp3 in tumor. TUNEL fluorescence staining was used to detect the apoptosis in the tumor. The radiopharmaceutical molecular probe 131I-atezolizumab showed high stability and in vivo biological activity. The treatment regimen adopted had a positive effect on the survival of tumor-bearing mice. 131I internal irradiation upregulated Caspase8 in tumor and ultimately inhibited solid tumor growth by activating apoptosis pathways. We also found a significant increase in the expression of NLRP3, which plays an important role in the pyroptosis pathway, in tumor. In summary, our data demonstrated that radiopharmaceuticals combined with immunotherapy affected tumor tissue by modulating relevant biological pathways, thereby achieving better antitumor effects compared with single therapy and providing new insights for promoting better patient prognosis and combination treatment strategies.

Stromal KITL/SCF promotes pancreas tissue homeostasis and restrains tumor progression.

Components of normal tissue architecture serve as barriers to tumor progression. Inflammatory and wound-healing programs are requisite features of solid tumorigenesis, wherein alterations to immune and non-immune stromal elements enable loss of homeostasis during tumor evolution. The precise mechanisms by which normal stromal cell states limit tissue plasticity and tumorigenesis, and which are lost during tumor progression, remain largely unknown. Here we show that healthy pancreatic mesenchyme expresses the paracrine signaling molecule KITL, also known as stem cell factor, and identify loss of stromal KITL during tumorigenesis as tumor-promoting. Genetic inhibition of mesenchymal KITL in the contexts of homeostasis, injury, and cancer together indicate a role for KITL signaling in maintenance of pancreas tissue architecture, such that loss of the stromal KITL pool increased tumor growth and reduced survival of tumor-bearing mice. Together, these findings implicate loss of mesenchymal KITL as a mechanism for establishing a tumor-permissive microenvironment.

Anti-HDGF antibody targets EGFR tyrosine kinase inhibitor-tolerant cells in NSCLC patient-derived xenografts.

Constitutively active mutant epidermal growth factor receptor (EGFR) is one of the major oncogenic drivers in NSCLC. Targeted therapy using EGFR tyrosine kinase inhibitor (TKI) is a firstline option in patients that have metastatic or recurring disease. However, despite the high response rate to TKI, most patients have a partial response, and the disease eventually progresses in 10 to 19 months. It is believed that drug-tolerant cells that survive TKI exposure during the progression-free period facilitate the emergence of acquired resistance. Thus, targeting the drug-tolerant cells could improve the treatment of NSCLC with EGFR mutations. We demonstrated here that EGFR mutant patient-derived xenograft (PDX) tumors responded partially to osimertinib despite near complete inhibition of EGFR activation. Signaling in AKT/mTOR and MAPK pathways could be reactivated shortly after initial inhibition. As a result, many tumor cells escaped drug killing and regained growth following about 35 days of continuous osimertinib dosing. However, when an antibody to hepatoma-derived growth factor (HDGF) was given concurrently with osimertinib, tumors showed complete or near-complete responses.There was significant prolongation of progression-free survival (PFS) of tumor-bearing mice as well. Immunohistochemistry and western blot analysis of tumors collected in the early stages of treatment suggest that increased suppression of the AKT/mTOR and MAPK pathways could be a mechanism that results in enhanced efficacy of osimertinib when it is combined with anti-HDGF antibody.

Nanomaterial-Mediated Delivery of MLKL Plasmids Sensitizes Tumors to Immunotherapy and Reduces Metastases.

Cancer immunotherapy has emerged as a promising approach for the induction of an antitumor response. While immunotherapy response rates are very high in some cancers, the efficacy against solid tumors remains limited caused by the presence of an immunosuppressive tumor microenvironment. Induction of immunogenic cell death (ICD) in the tumor can be used to boost immunotherapy response in solid cancers by eliciting the release of immune-stimulatory components. However, the delivery of components inducing ICD to tumor sites remains a challenge. Here, a novel delivery method is described for antitumor therapy based on MLKL (Mixed Lineage Kinase Domain-Like), a key mediator of necroptosis and inducer of ICD. A novel highly branched poly (β-amino ester)s (HPAEs) system is designed to efficiently deliver MLKL plasmid DNA to the tumor with consequent enhancement of immune antigen presentation for T cell responses in vitro, and improved antitumor response and prolonged survival in tumor-bearing mice. Combination of the therapy with anti-PD-1 treatment revealed significant changes in the composition of the tumor microenvironment, including increased infiltration of CD8+ T cells and tumor-associated lymphocytes. Overall, the HPAEs delivery system can enhance MLKL-based cancer immunotherapy and promote antitumor immune responses, providing a potential treatment to boost cancer immunotherapies.

Antibody targeting of anaerobic bacteria warms cold tumors and improves the abscopal effect of radiotherapy

BackgroundThe combination of immune checkpoint inhibitors with radiotherapy can enhance the immunomodulation by RT and reduce the growth of distant unirradiated tumors (abscopal effect); however, the results are still not very satisfactory. Therefore, new treatment options are needed to enhance this effect. Our previous study showed that the combination of Bifidobacterium (Bi) and its specific monoclonal antibody (mAb) could target and alleviate hypoxia at the tumor site and act as a radiosensitizer. In this study, we explored the anti-tumor efficacy of quadruple therapy (Bi + mAb and RT + αPD-1). The current study also aimed to probe into the complex immune mechanisms underlying this phenomenon.MethodsConstructed 4T1 breast and CT26 colon cancer tumor models. A comprehensive picture of the impact of constructed quadruple therapy was provided by tumor volume measurements, survival analysis, PET/CT imaging, immune cell infiltration analysis and cytokine expression levels.ResultsThe abscopal effect was further amplified in the “cold” tumor model and prolonged survival in tumor-bearing mice. Bi can colonized in primary and secondary tumors and direct the mAb to reach the tumor site, activate complement, enhance the ADCC effect and initiate the innate immune response. Then combined with αPD-1 and radiotherapy to stimulate adaptive immune response and synergize with cytokines to expand the immune efficacy and generate effective anti-tumor immune response.ConclusionsBi was used as an artificially implanted anaerobic target to cause a transient “infection” at the tumor, causing the tumor to become locally inflamed and “hot”, and at the same time, mAb was used to target Bi to enhance the local immune effect of the tumor, and then combined with radiotherapy and αPD-1 to amplify the abscopal effect in multiple dimensions. Therefore, the present study provided a new idea for the multipotent immune-activating function of antibody-targeted anaerobic bacteria for the RT treatment of extensively metastasized cancer patients.

TSG-6+ cancer-associated fibroblasts modulate myeloid cell responses and impair anti-tumor response to immune checkpoint therapy in pancreatic cancer

Resistance to immune checkpoint therapy (ICT) presents a growing clinical challenge. The tumor microenvironment (TME) and its components, namely tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs), play a pivotal role in ICT resistance; however, the underlying mechanisms remain under investigation. In this study, we identify expression of TNF-Stimulated Factor 6 (TSG-6) in ICT-resistant pancreatic tumors, compared to ICT-sensitive melanoma tumors, both in mouse and human. TSG-6 is expressed by CAFs within the TME, where suppressive macrophages expressing Arg1, Mafb, and Mrc1, along with TSG-6 ligand Cd44, predominate. Furthermore, TSG-6 expressing CAFs co-localize with the CD44 expressing macrophages in the TME. TSG-6 inhibition in combination with ICT improves therapy response and survival in pancreatic tumor-bearing mice by reducing macrophages expressing immunosuppressive phenotypes and increasing CD8 T cells. Overall, our findings propose TSG-6 as a therapeutic target to enhance ICT response in non-responsive tumors.

Elevated SLC3A2 associated with poor prognosis and enhanced malignancy in gliomas

The role of SLC3A2, a gene implicated in disulfidptosis, has not been characterized in gliomas. This study aims to clarify the prognostic value of SLC3A2 and its influence on glioma. We evaluated the expression of SLC3A2 and its prognostic importance in gliomas using publicly accessible databases and our clinical glioma samples and with reliance on Meta and Cox regression analysis approaches. Functional enrichment analyses were performed to explore SLC3A2's function. Immune infiltration was evaluated using CIBERSORT, ssGSEA, and single-cell sequencing data. Additionally, Tumor immune dysfunction and exclusion (TIDE) and epithelial-mesenchymal transition scores were determined. CCK8, colony formation, migration, and invasion assays were utilized in vitro, and an orthotopic glioma xenograft model was employed in vivo, to investigate the role of SLC3A2 in gliomas. Bioinformatics analyses indicated high SLC3A2 expression correlates with adverse clinicopathological features and poor patient prognosis. Upregulated SLC3A2 influenced the tumor microenvironment by altering immune cell infiltration, particularly of macrophages, and tumor migration and invasion. SLC3A2 expression positively correlated with immune therapy indicators, including immune checkpoints and TIDE. Elevated SLC3A2 was revealed as an independent risk element for poor glioma prognosis through Cox regression analyses. In vitro experiments showed that reduced SLC3A2 expression decreased cell proliferation, migration, and invasion. In vivo, knockdown of SLC3A2 led to a reduction in tumor volume and prolonged survival in tumor-bearing mice. Therefore, SLC3A2 is a prognostic biomarker and associated with immune infiltration in gliomas.

PLX038A, a long-acting SN-38, penetrates the blood-tumor-brain-barrier, accumulates and releases SN-38 in brain tumors to increase survival of tumor bearing mice

Central nervous system tumors have resisted effective chemotherapy because most therapeutics do not penetrate the blood-tumor-brain-barrier. Nanomedicines between ~ 10 and 100 nm accumulate in many solid tumors by the enhanced permeability and retention effect, but it is controversial whether the effect can be exploited for treatment of brain tumors. PLX038A is a long-acting prodrug of the topoisomerase 1 inhibitor SN-38. It is composed of a 15 nm 4-arm 40 kDa PEG tethered to four SN-38 moieties by linkers that slowly cleave to release the SN-38. The prodrug was remarkably effective at suppressing growth of intracranial breast cancer and glioblastoma (GBM), significantly increasing the life span of mice harboring them. We addressed the important issue of whether the prodrug releases SN-38 systemically and then penetrates the brain to exert anti-tumor effects, or whether it directly penetrates the blood-tumor-brain-barrier and releases the SN-38 cargo within the tumor. We argue that the amount of SN-38 formed systemically is insufficient to inhibit the tumors, and show by PET imaging that a close surrogate of the 40 kDa PEG carrier in PLX038A accumulates and is retained in the GBM. We conclude that the prodrug penetrates the blood-tumor-brain-barrier, accumulates in the tumor microenvironment and releases its SN-38 cargo from within. Based on our results, we pose the provocative question as to whether the 40 kDa nanomolecule PEG carrier might serve as a “Trojan horse” to carry other drugs past the blood-tumor-brain-barrier and release them into brain tumors.

Abstract PO-001: GPR65-low tumor clones reprogram macrophage via VEGF signaling and confer antigen-independent CAR-T resistance

Abstract Chimeric antigen receptor (CAR)-T cell therapies can be curative for CD19+ hematological malignancies, though are limited by frequent patient relapse and response failures. Here, we identify GPR65 as a tumor-specific determinant of responsiveness to CAR-T cell therapy. In patients and an immune competent mouse model of B cell acute lymphoblastic leukemia (B-ALL) response to CAR-T cell therapy, high GPR65 is associated with a complete tumor response. In mice, GPR65 knockout (GPR65 KO) tumors are resistant to CAR-T cell therapy in vivo, without antigen loss. Single-cell network analyses reveal that GPR65 deficiency remodels tumor interactions with host macrophages, partially through increasing tumor VEGFA. This leads to increased macrophage numbers and preferential M2 macrophage polarization. Either depletion of host macrophages or deletion of VEGFA from GPR65 KO tumors restores responsiveness to CAR-T cell treatment. Anti-VEGFA therapy in combination with CAR-T cell administration also prolongs the survival of GPR65 KO tumor-bearing mice. Our results indicate that tumor GPR65 expression is important for B-ALL responses to CAR-T cell therapy through its impact on the tumor microenvironment and supports new approaches to identify and overcome CAR-T cell resistance. Citation Format: Jayadev Mavuluri. GPR65-low tumor clones reprogram macrophage via VEGF signaling and confer antigen-independent CAR-T resistance [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-001.

ATRT-19. CELL SURFACE PROTEOME ANALYSIS OF ATRT IDENTIFIES CD44V6 AS A CAR TARGET

Abstract Atypical teratoid rhabdoid tumor (ATRT) is an uncommon, aggressive tumor affecting the brain and spinal cord primarily in children. Despite significant strides in understanding cancer biology through DNA and RNA sequencing, nearly 90% of ATRTs exhibit a crucial deletion that triggers rampant tumor growth. The absence of easily targetable genetic mutations in ATRT complicates the development of effective treatments, necessitating innovative strategies for therapy. Evading immune system detection is a key feature of cancer cells. In our study, we conducted an exhaustive, unbiased examination of 350 surface receptors through high-throughput multicolor flow cytometry on samples from ATRT surgeries, cell lines, and xenograft models derived from patients. By using a combination of antibodies targeting CD31, CD45, CD11b, CCR2, Cx3CR1, CD4, and CD8, we could identify immune cell populations within the tumors. Our findings revealed a heightened presence of CD44, CD146, CD59, CD151, and CD276 on the surface. Using qPCR we identified CD44V6 a variant isoform of the CD44 molecule, which is a cell surface glycoprotein involved in cell-cell interactions, cell adhesion, and migration. CD44 has multiple isoforms generated through alternative splicing, with CD44V6 being one of these variants. This specific isoform includes an additional variable exon (exon v6) in its extracellular domain. CD44v6-directed CAR-T cells effectively controlled tumor growth in multiple myeloma and solid tumors such as HNSCC, lung and ovarian adenocarcinomas. We developed CD44V6-directed CAR-T cells and investigated the capacity of Anti-CD44v6 CAR-T cells to execute cytotoxicity against ATRT tumor cells. We see significant cell killing by Anti-CD44v6 CAR-T cells in vitro and a significant increase in the survival of ATRT tumor-bearing mice.

IMMU-20. INNATE IMMUNE EVASION IN MYC-AMPLIFIED MEDULLOBLASTOMA

Abstract BACKGROUND While major advances have been made in improving the quality of life and survival of children with most forms of medulloblastoma (MB), those with MYC-driven tumors (Grp3-MB) still suffer significant morbidity and mortality. Here, we report a cell surface proteome analysis of Group3-MB cell lines after direct MYC inhibition to identify changes in surface proteins and sensitivity to macrophage-mediated phagocytosis. METHODS We had previously demonstrated the preferential activity of HDACi (CI994) in MYC-driven medulloblastoma. CI-994, showed significant cell viability reduction mediated by reduction in the MYC (mRNA and protein), induction of apoptosis in MYC-driven medulloblastoma. In this study, we directly inhibited MYC using MYCi975 and OMO-MYC and carried out a cell surface proteomic analysis on Group3-MB cell lines to reveal CD24 and CD59 as potential immune evasion markers. While CD24 is a potential don’t eat me signal, CD59 is a ubiquitously expressed cell-surface glycosylphosphatidylinositol-anchored protein that acts as an inhibitor to the membrane attack complex and protects cells from CDC. In certain tumors, CD59 expression is enhanced, posing a significant obstacle to treatment by hindering effective monoclonal antibody-induced CDC. We use inhibitors to CD24 and CD59 to confirm their role as innate immune evasion proteins in Group 3MB. RESULTS Meta-analysis of published datasets reveals CD24 and CD59 as strong prognostic indicators in Group3-MB as well as within the Group3g-subtype. Inhibition of MYC reduces surface levels of CD24, increasing macrophage-mediated phagocytosis. The use of a blocking anti-CD24 mAb significantly enhanced macrophage-mediated phagocytosis. Furthermore, a significant reduction in surface expression was observed in CD59. Blocking CD59 mAb significantly enhanced anti-CD47 mAb mediated phagocytosis. Combined treatment of anti-CD24 and anti-CD47 significantly enhanced the survival of Grp3-MB tumor-bearing mice.

Enhanced treatment of breast cancer brain metastases with oncolytic virus expressing anti-CD47 antibody and temozolomide

Limited therapeutic options are available for patients with breast cancer brain metastases (BCBM), and thus there is an urgent need for novel treatment approaches. We previously engineered an effective oncolytic herpes simplex virus-1 (oHSV) expressing a full-length anti-CD47 monoclonal antibody (mAb) with a human IgG1 scaffold (OV-αCD47-G1) that was used to treat both ovarian cancer and glioblastoma. Here, we demonstrate that the combination of OV-αCD47-G1 and temozolomide (TMZ) improve outcomes in preclinical models of BCBM. The combination of TMZ with OV-αCD47-G1 synergistically increased macrophage phagocytosis against breast tumor cells and led to greater activation of NK cell cytotoxicity. Additionally, the combination of OV-αCD47-G1 with TMZ significantly prolonged the survival of tumor-bearing mice when compared to TMZ or OV-αCD47-G1 alone. Combination treatment with the mouse counterpart of OV-αCD47-G1, termed OV-A4-IgG2b, also enhanced mouse macrophage phagocytosis, NK cell cytotoxicity, and survival in an immunocompetent model of mice bearing BCBM compared to TMZ or OV-A4-IgG2b alone. Collectively, these results suggest that OV-αCD47-G1 combined with TMZ should be explored in patients with BCBM.

The rationale of reprogramming TREM2+ macrophages for overcoming the resistance to immune checkpoint inhibitors in pancreatic adenocarcinoma.

e14649 Background: Pancreatic ductal adenocarcinoma (PDAC) resists to immune checkpoint inhibitors as single agents. TAMs expressing the triggering receptor expressed on myeloid cells (TREM2) protein were found to be a dominant macrophage population in PDAC; targeting TREM2 has been found to enhance the efficacy of immune checkpoint inhibitor-based therapies in preclinical studies of other malignancies. Here, we studied the role of targeting TREM2 in PDAC by testing the combination of non-depleting anti-TREM2 antibody and anti-PD-1 antibody in a mouse model of PDAC. We also examined the relationship between TREM2 expression and effector T cells in surgically resected human PDAC collected from a previous clinical trial treated with the neoadjuvant combination therapy of nivolumab (anti-PD-1 antibody), urelumab (anti-CD137 agonist antibody), and the pancreatic cancer GVAX vaccine. Methods: C57BL/6 mice were orthotopically implanted with KPC tumors. Anti-TREM2 was injected intraperitoneally (i.p.) once a week and anti-PD-1 was injected i.p. twice a week. Tumor volume was measured by ultrasound. TREM2 was stained on slides which were previously obtained from surgically resected PDACs following the neoadjuvant combination treatment of nivolumab, urelumab, and GVAX and stained for multiple immune cell markers with a multiplex staining-striping immunohistochemistry (IHC) technique. Results: Tumor growth post-tumor implantation was found to be significantly decreased by the anti-PD-1 and anti-TREM2 combination compared with anti-PD-1 alone, anti-TREM2 alone, or control treatment (p < 0.0001). Survival of tumor-bearing mice was significantly prolonged by the anti-PD-1 and anti-TREM2 combination when compared to anti-PD-1(p < 0.05), anti-TREM2(p < 0.05), and control (p < 0.01). Multiplex IHC analysis of human PDACs following neoadjuvant immunotherapy showed a higher density of granzyme B+CD8+ effector T cells correlated with higher density of TREM2+ cells (p = 0.0357). Conclusions: Our study demonstrates the anti-tumor efficacy of anti-TREM2 antibody in combination with an immune checkpoint inhibitor in mouse PDAC. However, the analysis of human PDAC following neoadjuvant immunotherapy suggests that TREM2 on TAMs may be activated following the activation of effector T cells. Taken together, our results suggest that TREM2+ TAMs should be reprogrammed instead of being depleted in the design of anti-TREM2-based therapy for PDAC.

Bioelectrocatalytic Activity of One-Dimensional Porous Pt Nanoribbons for Efficient Inhibition of Tumor Growth and Metastasis.

Designing and synthesizing one-dimensional porous Pt nanocrystals with unique optical, electrocatalytic, and theranostic properties are gaining lots of attention, especially to overcome the challenges of tumor recurrence and resistance to platinum-based chemotherapy. Herein, we represented an interesting report of a one-step and facile strategy for synthesizing multifunctional one-dimensional (1D) porous Pt nanoribbons (PtNRBs) with highly efficient therapeutic effects on cancer cells based on inherent electrocatalytic activity. The critical point in the formation of luminescent porous PtNRBs was the use of human hemoglobin (Hb) as a shape-regulating, stabilizing, and reducing agent with facet-specific domains on which fluorescent platinum nanoclusters at first are aggregated by aggregation-induced emission phenomena (AIE) and then crystallized into contact and penetration twins, as intermediate products, followed by shaping of the final luminescent porous ribbon nanomaterials, owing to oriented attachment association via the Ostwald ripening mechanism. From a medical point of view, the key strategy for effective cancer therapy occured via using low-dosage ethanol in the presence of electroactive porous PtNRBs based on intracellular ethanol oxidation-mediated reactive oxygen species (ROS) generation. The role of heme groups of Hb, as electrocatalytically active centers, was successfully demonstrated in both kinetically controlled anisotropic growth of NRBs for slowing down the reduction of Pt(II) followed by oligomerization of Pt(II)-Hb complexes via platinophilic interactions as well as electrocatalytic ethanol oxidation for therapy. Interestingly, hyaluronic acid-targeted (HA) Hb-PtNRB in the presence of low-dose ethanol caused extraordinary arrest of tumor growth and metastasis with no recurrence even after the treatment course stopped, which caused elongation of tumor-bearing mice survival. HA/Hb-PtNRB was completely biocompatible and exhibited high tumor-targeting efficacy for fluorescent imaging of breast tumors. Therefore, the synergistic electrocatalytic activity of PtNRBs is presented as an efficient and safe cancer theranostic method for the first time.