Survival Of Mice Research Articles

Small but mighty: targeted antifungal liposomes of a smaller size are superior in treating cryptococcal meningitis

ABSTRACT Cryptococcal meningoencephalitis (CME) is deadly. CME is responsible for 19% of deaths in AIDS patients, and its global mortality is greater than 60%. The recommended CME therapy requires amphotericin B (AmB), a fungicidal drug targeting fungal ergosterol. AmB also binds to the host’s cholesterol and is highly toxic. Liposomal AmB (AmB-LLs), relative to deoxycholate-solubilized AmB, has lower toxicity and longer tissue retention, but it requires high doses for treatment and its efficacy in treating CME remains unsatisfactory. To improve the effectiveness of AmB-LLs, we previously developed DectiSomes-targeted AmB-LLs decorated with host dectins that recognize fungal polysaccharides. DectiSomes, relative to untargeted AmB-LLs, modestly improve efficacy against systemic cryptococcosis, in contrast to the drastic improvement observed in candidiasis or aspergillosis models. We speculated that limited tissue penetration of the regular-sized DectiSomes might have contributed to the modest improvement in treating systemic cryptococcosis. Here, we discovered that DectiSomes of a smaller size (~50 nm), compared with DectiSomes of the regular size (~100 nm) or untargeted AmB-LLs of either size, had a much better capability in reducing cryptococcal burden of various organs including the brain and in prolonging the survival of mice with systemic cryptococcosis. The performance of small DectiSomes was far superior to all other groups at two different doses of AmB tested. Furthermore, no kidney toxicity was observed in any of the treatment regimens tested. Taken together, our findings indicate that small DectiSomes can be a powerful antifungal delivery platform to drastically improve therapies against the deadly CME. IMPORTANCE Systemic cryptococcosis is fatal even with antifungal interventions. The most effective drug against this disease is amphotericin B (AmB). However, AmB is highly toxic as it binds to fungal ergosterol and also mammalian cholesterol. Liposomal AmB was introduced to the clinic in 1990s because it showed reduced toxicity and longer retention in various organs. However, the dose of AmB required for treatment using liposomal formulation is high and the outcome is far from satisfactory. In our previous work, we generated DectiSomes, dectin-decorated liposomes loaded with AmB that more effectively deliver the drug to the pathogen and enhance antifungal efficacy. However, the improvement in treating systemic cryptococcosis, compared with candidiasis and aspergillosis, is modest. Here, we generated DectiSomes that are half their regular size to improve tissue penetration. We discovered that small DectiSomes are superior in reducing fungal burden in various organs including the brain and in prolonging animal survival.

Abstract PR010: TRPV1+ sensory innervation as a novel driver of ovarian cancer progression

Abstract Introduction: A primary challenge currently faced in ovarian cancer (OVCA) treatment is that patient prognoses remain poor despite current therapeutic interventions. Overall survival for OVCA patients with advanced disease is <30%, and most patients recur within 5 years. As such, there is a critical need for new therapies that can overcome OVCA chemoresistance and produce meaningful increases in patient survival. Recent efforts that have targeted the tumor microenvironment (TME), such as immune checkpoint inhibitors, have been successful in some cancers but have limited therapeutic benefit when used to treat OVCA. Thus, new TME targeting therapies must be developed to effectively treat OVCA patients. A novel TME component ripe for therapeutic targeting are the nerve fibers that infiltrate tumors. Recent studies in several cancers have shown that tumor innervation can promote tumor growth/metastasis. However, in ovarian cancer the role of innervation in promoting cancer progression remains a gap in knowledge. Here we show that Transient Receptor Potential Cation Channel Subfamily V Member 1 (TRPV1)+ sensory innervation plays a significant role in driving ovarian cancer progression. Methods: We performed IHC staining to determine the extent of TRPV1+ nerve infiltration in OVCA vs normal reproductive tissue in patients. We examined the ability of OVCA cells to promote tumor innervation using a neurite outgrowth assay. We utilized both syngeneic and patient derived xenograft (PDX) mouse models of ovarian cancer metastasis, where mouse or human OVCA cells were injected intraperitoneally, to explore the functional role of TRPV1+ sensory nerves in OVCA. TRPV1+ sensory nerves were ablated genetically using TRPV1 driven expression of diphtheria toxin alpha (DTA) or chemically using resiniferatoxin. TRPV1+ sensory nerves were activated pharmacologically using capsaicin. Results: Analysis of patient samples showed that TRPV1+ sensory innervation is much higher in ovarian tumors vs benign reproductive tissue. We found that conditioned media from OVCA cells was able to promote neurite outgrowth of dissociated mouse sensory neurons. Ablation of TRPV1+ sensory nerve fibers in vivo caused reduced tumor burden and prolonged survival in our syngeneic and PDX OVCA mouse models. Stimulation of TRPV1+ sensory nerves with capsaicin accelerated OVCA growth and decreased survival in tumor bearing mice. Conclusions: Our results establish TRPV1+ sensory innervation as a novel driver of OVCA growth/metastasis and a potential therapeutic target for OVCA treatment. Significance to tumor microenvironment field: Our data add to a growing body of evidence that sensory nerves are pro-tumorigenic in multiple cancer types and could be therapeutically targeted for cancer treatment. Citation Format: Matthew Knarr, Katherine Cummins, Dusan Racordon, Hunter Reavis, Timothy Lippert, Ryan Hausler, Priyanka Rawat, Jamie Moon, Dave S.B. Hoon, Roger Greenberg, Paola Vermeer, Ronny Drapkin. TRPV1+ sensory innervation as a novel driver of ovarian cancer progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR010.

Abstract C030: Timing of melanoma brain metastasis seeding dictates the immunomodulatory effect and efficacy of systemic anti-CD40 agonist therapy

Abstract Introduction: Melanoma brain metastasis (MBM) affects nearly 50% of patients with advanced melanoma. Despite progress in systemic therapies, particularly immune checkpoint inhibitors, intracranial responses remain highly variable. The CD40/CD40L axis and its pleiotropic effects on the anti-tumoral immune response has the potential to improve MBM treatment. Our aim was to investigate whether the timing of MBM seeding, in a two-site mouse model of MBM, impacts the intracranial efficacy of anti-CD40 agonism (aCD40). Methods: 2 x 105 B16F1-cOVA murine melanoma cells were subcutaneously (s.c.) injected in the right flank in C57BL/6 mice (5F, 5M). After 3 days, 1 x 105 B16F1-cOVA cells were intracerebrally (i.c.) injected into the right striatum under stereotaxic guidance (early MBM’ cohort). A separate cohort of C57BL/6 mice (5F, 5M) had s.c. injection of B16F1-cOVA cells, followed by an i.c. injection of B16F1-cOVA cells after 5 days (‘late MBM’ cohort). In both experimental cohorts, the mice were randomly allocated to receive either an aCD40 antibody or an isotype IgG antibody (control) intraperitoneally on days 4 (D4) and 7 (D7) post-i.c. injection (D0). S.c. tumor volume and survival data were serially collected. Separately, tumor-bearing mice treated with either aCD40 or IgG (n = 4 per early/late MBM groups) were euthanized day 9 (D9) post-i.c. injection and tissues (blood, brain tumor [BT], and flank tumor [FT]) were collected for immune profiling by spectral flow cytometry. Results: The early MBM cohort showed a significant s.c. tumor response to aCD40 therapy with a mean D9:D4 s.c. tumor volume ratio of 2.81 (SD±2.05) vs 16.51 (±13.10) in the IgG group (unpaired t-test; p=0.0497). The late MBM cohort showed no significant difference in s.c. tumor response between aCD40 or IgG (D9:D4 ratio 1.52±1.00 vs 1.20±0.83; p=0.5999). Survival increased in early MBM following aCD40 vs IgG (median 20 days vs 13 days; logrank test, p=0.0078), which was not replicated in late MBM (median 21 days vs 19 days; p=0.7717). Systemic aCD40 led to reduced circulating B cells, most expressing CD11b, and increased monocytes, compared to IgG treatment. In the BT, there were higher infiltration of CD8 T cells (normalized to microglia), including OVA+ CD8 T cells, and CD4 T cells in early MBM treated with aCD40 vs IgG. Systemic aCD40 induced more infiltrating SIRP1a+ cDC2 cells than XCR1+ cDC1 cells and vice versa in the IgG group, in both the BT and FLT. Meanwhile, infiltrating B cells were predominantly CD11b+ in aCD40 groups in the BT, regardless of MBM timing. Conclusion: Systemic aCD40 therapy improved survival of MBM-bearing mice, with a shorter interval between s.c. and i.c. implantation; an effect abrogated by later MBM seeding. Systemic aCD40 therapy induced specific changes in the BT infiltrative immune profile, which may explain the distinct response between the early and late BM cohorts. These findings have implications on the experimental modelling of MBM and provide insight into variability of MBM response to immunomodulating therapies. Citation Format: Vinton W. T. Cheng, Victoria R. Breza, Beata Chertok, Natasha D. Sheybani, Timothy N. J. Bullock, Richard J. Price. Timing of melanoma brain metastasis seeding dictates the immunomodulatory effect and efficacy of systemic anti-CD40 agonist therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C030.

Abstract C039: The impact of antiangiogenic therapy on blood vessels and CD8 infiltration in renal cancer metastases is space, time, site, dose, and drug dependent

Abstract Clear cell renal cell carcinoma (ccRCC) is the most prevalent renal neoplasm, with bone as a major site for distant spread in 35% to 40% of the patients. These metastases are typically osteolytic, resistant to treatments, hence result in a variety of skeletal-related complications strongly contributing to mortality. A key feature of ccRCC is the loss of function of the von Hippel–Lindau (VHL) protein leading to extensive angiogenesis. Consequently, different anti-angiogenic tyrosine kinase inhibitors (TKIs) are used as the first or subsequent line of treatment for these patients as single agents or in combination with immune checkpoint inhibitors. However, limited data about their efficacy in bone metastasis is available, and their impact on the immune infiltrate, including effector CD8 cells, is unclear. We hypothesized that TKIs, by inhibiting angiogenesis, significantly reprogram the microenvironment and limit immune infiltration in space, time, dose, and drug dependent fashion. To test these hypotheses, we developed an immunocompetent mouse model of bone metastasis by orthotopical intratibial implantation of luciferase-GFP-positive mouse RENCA-VHL- cell line. Interestingly, intratibial injections gave rise to metastases in lungs, creating a unique opportunity to study two metastatic sites. Three different TKIs currently used in patients were tested and compared to control: axitinib (A), cabozantinib (C), and lenvantinib (L). Outcomes were monitored by macroscopic bioluminescence detection in vivo, 3D multiparametric spatial analysis of bones and lungs, in vivo, and ex vivo. Compared to control-treated mice, high doses of A, C, and L significantly reduced tumor progression in both tumor sites. Even though these TKIs are clinically considered equal, significant differences in their efficacy were noted. Spatial analysis of tumor cells and blood vessels showed a significant decrease in blood vessels formation in both an organ- and TKI-specific pattern over time. Additionally, the reduction of neo angiogenesis in treated tumors directly limited the infiltration of CD8 cells, which localized at the interface with tumor free bone/lung tissue. Interestingly, TKI dose reduction significantly improved CD8 infiltration, while controlling tumor size. TKI withdrawal, instead, led to rapid progression of tumor in both sites and significantly shortened mice survival. In conclusion, we established bone and lung metastatic models of ccRCC and characterized and compared the effects of three antiangiogenic TKIs on angiogenesis and CD8 infiltration. TKIs reduced tumor growth and progression, because of blood vessel formation inhibition. Furthermore, TKI treatment reduced the infiltration of effector T cells in tumors in a dose-dependent manner, an outcome that can have a major impact when TKIs are combined with immunotherapy, a preferential therapeutic regimen for ccRCC patients. Overall, we believe our studies provide important insights and efficacy predictions for innovative therapeutic applications in ccRCC bone metastatic patients. Citation Format: Stefan Maksimovic, Nina Costanza Boscolo, Ludovica La Posta, Matthew T. Campbell, Eleonora Dondossola. The impact of antiangiogenic therapy on blood vessels and CD8 infiltration in renal cancer metastases is space, time, site, dose, and drug dependent [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C039.

A PSMA-targeted Tri-specific Killer Engager enhances NK cell cytotoxicity against prostate cancer.

Natural killer (NK) cell tumor infiltration is associated with good prognosis in patients with metastatic castration-resistant prostate cancer (mCRPC). NK cells recognize and kill targets by a process called natural cytotoxicity. We hypothesized that promoting an antigen-specific synapse with co-activation may enhance NK cell function in mCRPC. We describe a Tri-specific Killer Engager (TriKE) construct that engages with the activating receptor CD16 on NK cells, prostate-specific membrane antigen (PSMA) on mCRPC cells, and has an interleukin (IL)-15 moiety that is essential for NK cell survival, proliferation, and priming. We show that the PSMA TriKE specifically binds to PSMA-expressing cells and significantly enhances expansion, degranulation and cytokine production of NK cells derived from healthy donors or prostate cancer patients. Bystander killing of PSMA-negative was also achieved with PSMA TriKE treatment when co-cultured with PSMA-positive cells, suggesting potential PSMA TriKE benefit in controlling tumor antigen escape. When tested under physiologic conditions recapitulating the mCRPC tumor microenvironment (TME), NK cells treated with PSMA TriKE and prolonged exposure to hypoxia or MDSCs maintained their potent function while IL-15 treated NK cells showed greatly impaired cytotoxicity. Finally, in vivo testing of PSMA TriKE showed improved tumor control and survival of mice as compared to IL-15 and untreated control groups. In conclusion, PSMA TriKE demonstrates potential as a new therapy for advanced prostate cancer by providing additional signals to NK cells to maximize their anti-tumor potential in prostate cancer, especially in the setting of a hostile TME.

Inducible global knockout of surfeit locus protein 4 in adult mice results in hypolipidemia, intestinal lipid accumulation, liver injury, and increased mortality.

Surfeit locus protein 4 (SURF4) acts as a cargo receptor to mediate endoplasmic reticulum export of various cargos. We have shown that SURF4 is essential for secretion of hepatic very low-density lipoprotein and intestinal chylomicron. Knockdown of hepatic Surf4 also significantly reduces the development of atherosclerosis and liver fibrosis without causing overt liver damage. However, constitutive global Surf4 knockout results in embryonic lethality. To further understand the physiological role of Surf4, we generated tamoxifen-inducible global Surf4 knockout mice. We found that conditional knockout of Surf4 in adult mice (Surf4ig-ko) significantly reduced mouse body weight. Male and female Surf4ig-ko mice died approximately 30 and 50 days after tamoxifen administration, respectively. Triglyceride secretion and serum levels of total cholesterol, triglycerides, free fatty acids, apolipoprotein B-100, and apolipoprotein-48 were significantly reduced in Surf4ig-ko mice compared with Surf4flox mice. Proteomics analysis of mouse serum samples revealed 308 proteins with significantly altered expression in Surf4ig-ko mice that have unique functions and are involved in various biological processes. In addition, Surf4ig-ko mice exhibited lipid accumulation in the intestine but not in the liver. However, in Surf4ig-ko mice, liver weight was significantly reduced, and serum transaminase activity was significantly increased, indicating liver damage. Therefore, SURF4 is essential for survival in adult mice, suggesting that the therapeutic use of SURF4 requires precise tissue/cell type-specific targeting.

Abstract B002: A therapeutically targetable positive feedback loop between long noncoding RNA-HLX-2-7, HLX, and MYC that promotes childhood cancer group 3 medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor. MB is categorized into four main molecular subgroups: wingless pathway (WNT)-activated, sonic hedgehog pathway (SHH)-activated, group 3 (G3), and group 4 (G4). Although the driver pathways for G3 and G4 MB are unclear, most of these tumors are characterized by c-Myc and MYCN signatures, respectively. Recent studies suggest that long non-coding RNAs (lncRNAs) contribute to medulloblastoma formation and progression. We have identified lncRNA, lnc-HLX-2-7, as an oncogene and a potential therapeutic target in group 3 (G3) medulloblastomas. lnc-HLX-2-7 RNA specifically accumulates in the promoter region of its host coding gene HLX, a sense overlapping gene of lnc-HLX-2-7, which activates HLX expression by recruiting multiple factors, including enhancer elements. RNA sequencing and chromatin immunoprecipitation reveal that HLX binds to and activates the promoters of several oncogenes, including TBX2, LIN9, HOXM1, and MYC. Intravenous treatment with cerium oxide nanoparticle–coated antisense oligonucleotides targeting lnc-HLX-2-7 (CNP-ASO-lnc-HLX-2-7) and lipid nanoparticle-coated HLX (LNP-si-HLX) inhibits tumor growth by 40-50% in an intracranial medulloblastoma xenograft mouse model (p<0.01). Combining nanoparticle-coated lnc-HLX-2-7 and HLX with standard-of-care cisplatin further inhibits tumor growth and significantly prolongs mouse survival compared with monotherapy (p<0.01). Thus, the lnc-HLX-2-7–HLX–MYC axis is important for regulating G3 medulloblastoma progression, providing a strong rationale for using lnc-HLX-2-7 and HLX as therapeutic targets for G3 MBs. Citation Format: Ranjan R Perera. A therapeutically targetable positive feedback loop between long noncoding RNA-HLX-2-7, HLX, and MYC that promotes childhood cancer group 3 medulloblastoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B002.

Abstract B004: Ovarian cancer long intergenic noncoding RNA 1 is a potential therapeutic target in advanced ovarian cancer by promoting glycolytic pathway and hypoxic response

Abstract Ovarian cancer (OvC) is often diagnosed as an advanced disease because symptoms do not develop at earlier stages. Besides current treatments, alternative efficient therapies for OvC need to be established. We have previously identified that a novel long intergenic noncoding RNA (lincRNA) originated from chromosome 10q21 was predominantly expressed in clinical ovarian cancer tissues, designated as ovarian cancer lincRNA 1 (OIN1). Silencing of OIN1 in OvC cell lines substantially exhibited the inhibition of in vitro and in vivo cell proliferation, by upregulating apoptosis-related genes. In the present study, we aimed to evaluate whether OIN1 can be a therapeutic target in advanced OvC and how OIN1 drives the progression of OvC. We established long-term culturable spheroid cultures of OvC patient-derived cells (OvC-PDCs) from ascites of OvC patients as an advanced OvC model. The OvC-PDC spheroid cultures abundantly expressed OIN1 and stemness-related genes. OIN1-targeted siRNAs could suppress the spheroid growth and upregulate apoptosis-related genes such as RASSF5 and ADORA1 in OvC-PDC cultures. In a bloody ascites-generating xenograft in vivo model of mice intraperitoneally injected with OvC-PDC, OIN1-targeted siRNA treatment engineered by drug delivery system alleviated ascites production and prolonged the mice survival time. Based on transcriptomic analysis of OIN1 silencing in OvC-PDC cultures, we found that OIN1 inhibition exerts the downregulation of genes involved in glycolytic pathway and hypoxic response. We further demonstrated that OIN1 modulates HIF1a-mediated transcriptional regulation in OvC- PDCs. The present findings provide promising evidence that OIN1 can be a potential therapeutic target for advanced OvC. Citation Format: Kuniko Horie, Wataru Sato, Kazuhiro Ikeda, Satoshi Inoue. Ovarian cancer long intergenic noncoding RNA 1 is a potential therapeutic target in advanced ovarian cancer by promoting glycolytic pathway and hypoxic response [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B004.

Loop33 × 123 CAR-T targeting CD33 and CD123 against immune escape in acute myeloid leukemia.

Immunotherapy, such as chimeric antigen receptor T (CAR-T) cells targeting CD33 or CD123, has been well developed over the past decade for the treatment of acute myeloid leukemia (AML). However, the inability to sustain tumor-free survival and the possibility of relapse due to antigen loss have raised concerns. A dual targeting of CD33 and CD123 is needed for better outcomes. Based on our previously constructed CD33 and CD123 monovalent CAR-T, Loop33 × 123 and Loop123 × 33 CAR-T were constructed with molecular cloning techniques. All CAR-T cells were generated by lentivirus transduction of T cells from healthy donors. Phenotype detection was evaluated on day 7 concerning activation, exhaustion, and subtype proportions. Coculture killing assays were conducted using various AML cell lines and primary AML cells. Degranulation and cytokine secretion levels were detected by flow cytometry. Cell-derived xenograft models were established using wild-type Molm 13 cell lines, or a mixture of Molm 13-KO33 and Molm 13-KO123 cells as an ideal model of immune escape. By monitoring body weight and survival of tumor-bearing mice, Loop33 × 123 and Loop123 × 33 CAR-T cells were further assessed for their efficacy in vivo. In vitro study, our results demonstrated that Loop33 × 123 CAR-T cells could efficiently eliminate AML cell lines and primary AML cells with elevated degranulation and cytokine secretion levels. Compared with our previously constructed monovalent CD33 or CD123 CAR-T cells, Loop33 × 123 CAR-T cells showed superior advantages in an immune escape model. In vivo studies further confirmed that Loop33 × 123 CAR-T cells could effectively prolong the survival of mice without significant toxicity. However, Loop123 × 33 CAR-T cells failed to show the same effects. Furthermore, Loop33 × 123 CAR-T cells efficiently circumvented potential immune escape, a challenge where monovalent CAR-T cells failed. Loop33 × 123 CAR-T targeting CD33 and CD123 could efficiently eliminate AML cells and prolong survival of tumor-bearing mice, while addressing the issue of immune escape.

In Vitro and In Vivo Antitumor Activity of Lophocereus marginatus (DC.) S. Arias & Terrazas Endophytic Aspergillus versicolor and Metarhizium anisopliae Extracts Against the Murine Lymphoma L5178Y-R

Cancer belongs to a group of diseases characterized by uncontrolled cell growth. The search for new effective treatments for cancer has led to the discovery of different molecules from plants, bacteria, and fungi with pharmacological use. Plant endophytic fungi are large producers of metabolites with antitumor properties. We aimed to evaluate the in vitro and in vivo antitumor potential of extracts from Lophocereus marginatus endophytic fungi. We obtained ethyl acetate and hexane extracts from the L. marginatus endophytes Metarhizium anisopliae and Aspergillus versicolor and evaluated their antitumor activity against murine L5178Y-R lymphoma cells and human peripheral blood mononuclear cells, using the 3-(4,5-dimethylthiazol-2-yl)-2-diphenyltetrazolium bromide reduction colorimetric technique. M. anisopliae and A. versicolor ethyl acetate extracts showed IC50 values of 9.168 ± 1.21 μg/mL and 13.51 ± 1.62, respectively, and selectivity indices > 30. We also observed that the maximum tolerated dose (100 mg/kg) of ethyl acetate extracts and the vehicle in BALB/c mice did not cause hepatotoxicity. In addition, we evaluated the effects of ethyl acetate extracts on survival and tumor volume in the L5178Y-R lymphoma tumor model. An increase in survival (17 d) was observed in mice treated with A. versicolor extract. Furthermore, it did not increase tumor volume during 10 d, as compared with the control groups without treatment, vehicle, and M. anisopliae extract, which had a maximum survival of 10 d. A. versicolor ethyl acetate extract showed in vitro and in vivo antitumor activity against lymphoma L5178Y-R, increasing mice survival.

Abstract 4134426: Targeting the TRIM29-PERK signaling axis for treating viral myocarditis

Introduction: Viral myocarditis is an inflammatory disease of the myocardium that significantly contributes to sudden death in children and young adults. The COVID-19 pandemic underscores the critical need to understand the pathogenesis and develop effective treatment strategies for viral myocarditis. Methods: To investigate the role of the novel E3 ligase TRIM29 in viral myocarditis, we used wild-type and TRIM29 knockout mice infected with coxsackievirus B3 (CVB3) and encephalomyocarditis virus (EMCV), to induce the myocarditis. Additionally, wild-type mice infected with CVB3 were treated with the protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibitor GSK2656157 or a DMSO control to evaluate potential therapeutic interventions. Mice survival and heart function were monitored using transthoracic echocardiography, and hearts were harvested for histological and immunohistochemical analysis. Techniques including real-time PCR, western blotting, co-immunoprecipitation, enzyme-linked immunoassay, flow cytometry, over-expression, and knockdown were employed to elucidate the pathogenic mechanisms by which TRIM29 regulates the endoplasmic reticulum stress response after viral infection. Results: We found that TRIM29 was highly induced by cardiotropic viruses, including CVB3 and EMCV, and promoted PERK-mediated endoplasmic reticulum (ER) stress, apoptosis, and reactive oxygen species (ROS) responses, which restricted viral replication in cardiomyocytes in vitro . TRIM29 deficiency protected mice from viral myocarditis by enhancing cardiac antiviral functions and reducing PERK-mediated inflammation and immunosuppressive cells in vivo . Mechanistically, TRIM29 interacted with PERK to catalyze the SUMOylation of PERK, maintaining its stability and thereby promoting PERK-mediated signaling pathways. Furthermore, we demonstrated that the PERK inhibitor GSK2656157 mitigated viral myocarditis by disrupting the TRIM29-PERK interaction, which improved cardiac function, enhanced cardiac antiviral responses, and reduced inflammation and immunosuppressive cells in vivo . Conclusions: Our findings provide the first evidence that the TRIM29-PERK signaling axis can be targeted for the treatment of viral myocarditis. This suggests that targeting the TRIM29-PERK axis could effectively mitigate viral myocarditis and associated cardiovascular diseases.

Abstract 4133460: VCAM1 contributes to pathogenesis during influenza-induced exacerbation of atherosclerosis.

Background and hypothesis: Influenza is a significant public health and economic threat around the world. Although pneumonia is the most common complication associated with influenza, there are several clinical reports demonstrating increased risk for cardiovascular disease. Influenza infection induces interferons, proinflammatory cytokines and chemokines, and recruits’ macrophages and neutrophils to control the virus. However, an excessive influx of innate immune cells and dysregulated production of inflammatory mediators results in pathological responses during influenza infection. Studies have shown that influenza infection correlates with increased incidence of myocardial infarction. Atherosclerosis is the most known cause of ischemic heart diseases and stroke. Vascular cell adhesion molecule-1 (VCAM1) has been shown to promote adhesion of monocytes and promotes atherosclerosis. In this study, we hypothesize that VCAM1 plays a role in exacerbation of atherosclerosis during influenza infection. Methods: High-Fat diet (HFD)-fed Apoe -/- mice were infected with influenza A/PR/8/34 (H1N1) and weight loss, survival rate, and gene expression of vascular endothelial adhesion molecules, inflammatory cytokines and chemokines were measured. HFD-fed Apoe -/- mice were infected with influenza, and treated with anti-VCAM1 antibody, and weight loss and cellular responses were measured. Results and conclusions: Increased weight loss and decreased survival of mice were observed in response to influenza infection in HFD-induced atherosclerosis in Apoe -/- mice. Further, the expression of VCAM1, and the levels of IL-6, CCL2, CCL3, CCL5 were significantly increased in aorta in Apoe -/- mice when compared to PBS-treated controls. Increased survival, decreased weight loss, decreased lesion area, decreased frequency of CD11b + F4/80 + Ly6C + , CD4 + RORgt + cells and increased frequency of CD4 + FoxP3 + Treg cells were observed in aorta in response to antibody mediated VCAM1 neutralization. These results suggest that VCAM1 plays a pathological role in influenza-induced exacerbation of atherosclerosis.

Abstract 4136204: Clearing Senescent Cells Improves Mouse Survival Rate Post Myocardial Infarction through Alteration of Cardiomyocyte and Immune Cell Subpopulations

Introduction: Cellular senescence often involves a p16-pathway, and p16 overexpression is a hallmark of senescent cells. The role of cellular senescence in myocardial infarction (MI) and any mediating mechanisms remain unclear. Aims: To investigate the effect of p16 + cell clearance on survival post MI and elucidate underlying mechanisms. Methods: We utilized INK-ATTAC transgenic mice, in which p16 + cells undergo targeted apoptosis upon exposure to AP20187 (AP). Sham and MI mice were treated with AP or vehicle (V) twice-weekly for one month, starting 3-4 hours post-MI. Survival rate improvement post MI in the AP group (Fig A, P<0.001) prompted us to evaluate effects of senescent cell clearance at the cardiac cellular level and its involvement in MI-associated pathways at 3 days post-MI. The infarct and peri-infarct areas were collected and snap-frozen. Nuclei were extracted with modified gentleMACS methods; libraries were constructed using 10X Genomics. Standard software (CellRanger, Seurat) were employed for single-nuclei-RNA-seq data analysis. Results: Cardiomyocytes (CMs) showed 5 distinct subsets. CM subclusters 1 and 2 showed enrichments of genes characteristic of infarct boarder zones, while most sham CMs were found in subcluster 0, reflecting healthy-CMs. AP-treated mice exhibited a higher proportion of healthy CMs (Fig B). Differentially expressed genes (DEGs) between V and AP group were enriched in the healthy CMs, suggesting that clearance of p16 + cells improves the status of CMs post MI (Fig C). Immune-cell profiling showed that by far the largest number of DEG for AP vs vehicle were expressed in the (arginase-1) Arg1 macrophage subgroup (Fig D). The percentage of Arg1 macrophages decreased in the AP group (FDR=0.059) (Fig E). We noted significant enrichment in the hypoxia and cardiac rupture pathways in Arg1 macrophages (Fig F), correlating with the high death rate from myocardial rupture in V-mice. Conclusion: These results suggest a significant role of p16 + senescent cells in post-MI mortality, potentially through effects on cardiomyocytes and a specific macrophage subpopulation, Arg1 macrophages.

DDDR-38. TRANSLATIONAL STEPS OF THE ANTITUMOR PEPTIDE TAT-CX43266-283 AS A TREATMENT FOR GLIOBLASTOMA: EGFR AS A TREATMENT RESPONSE BIOMARKER AND RESULTS OF COMBINATION THERAPY IN A PRECLINICAL RESECTION MODEL

Abstract Glioblastomas (GBM) are the most malignant primary brain tumors, and they remain incurable. Despite the current standard of care (surgical resection of the tumor and administration of chemotherapy and radiotherapy) some GBM stem cells (GSCs) remain in the brain parenchyma causing tumor recurrence. The Src-inhibitory peptide TAT-Cx43266-283 has shown promising antitumor results in GBM preclinical models, reducing GSC viability and increasing survival in GBM-bearing mice. Here we have investigated some of the main gaps to overcome when translating preclinical results to the clinic, to promote the progress of TAT-Cx43266-283 as a clinical therapy to fight GBM. First, we investigated biomarkers of treatment response to design a more targeted therapy. We assessed cell viability after treatment with TAT-Cx43266-283 in 13 patient-derived GSC lines and we found a stronger treatment response in those GSCs that had alterations in the Epidermal Growth Factor Receptor (EGFR). This was further confirmed in several murine GBM lines. Importantly, we found that treatment with TAT-Cx43266-283 was more effective in vitro than temozolomide or the classical EGFR inhibitor, erlotinib. Moreover, we showed that EGFR participates in TAT-Cx43266-283 response together with Src, by decreasing EGFR and EGFRvIII activity in some GSCs and increasing survival in mice bearing tumors with EGFR alterations. Next, we studied the effect of TAT-Cx43266-283 in a clinical context by administering it in a murine model of tumor resection. We analyzed the histopathology of this GBM model, uncovering histological features typical of human GBM. We found that resection alone improved, although not significantly, GBM-bearing mice survival. However, tumors that regrew after resection exhibited more aggressive features. Importantly, the combination of tumor resection and TAT-Cx43266-283 achieved better survival outcomes and showed reduced invasive features. Altogether, these results serve as a base for future clinical investigations and support the therapeutic potential of TAT-Cx43266-283 as a treatment for GBM.

IMMU-44. EXTRINSIC ELECTRICAL MODULATION OF THE GLIOBLASTOMA TUMOR MICROENVIRONMENT

Abstract BACKGROUND Approximately 30 to 50% of patients with glioblastoma (GBM) present with glioma-related epilepsy (GRE). Despite morbidity associated with seizures, GRE constitutes an independent positive predictive predictor for survival in GBM. Our prior results showed that repeated seizure induction through extrinsic electrical modulation (EEM, a technique in which an electrical stimulus sufficient to produce a therapeutic seizure is applied) slows intracranial tumor growth and prolongs overall survival in immunocompetent CT-2A glioma-bearing mice. However, the overall survival advantage following EEM treatment was diminished in immunocompromised mice bearing human patient derived xenografts. We hypothesized that EEM induces immune-related tumor microenvironment (TME) changes that prolong survival in mice. METHODS C57BL/6J mice were orthotopically implanted with CT-2A xenografts and subjected to EEM of 2.0 millicoulombs or sham treatment. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed at 3 and 24 hours to determine mRNA level of inflammatory cytokines. Immunohistochemistry (IHC) and bulk RNA sequencing analysis were utilized to assess CD3+ T-cell infiltration and identify immune-related secreted genes, respectively. Flow cytometry at 3 and 96 hours was utilized to characterize tumor-infiltrating immune cell populations. Samples were subjected to Percoll gradient enrichment of immune cell populations and stained for extracellular (CD45, CD4, CD8, CD11b, F4/80, CD80, Ly6G, Ly6C, and CX3CR1) and intracellular (TNF-a, Il-1b, CCL2, and Arg1) antigens. RESULTS RT-qPCR showed an increase in pro-inflammatory cytokines TNF-α (P = 0.0087) and IL-1β (P = 0.000349). IHC analysis demonstrated an increase in intratumoral CD3+ T-cells in EEM-treated samples (P = 0.0171). From 1855 upregulated genes, 3 immune-related secreted genes (C1QTNF4, FAM19A4, and GREM2) were identified. Flow cytometric analysis showed a significant increase in CD11b+, CX3RC1+ microglia (P = 0.0306) and CD45+, CD11b+ tumor-associated myeloid cells (TAMCs) (P = 0.0192) at 3 hours post-EEM. CONCLUSIONS EEM treatment promotes an inflammatory TME with increased intratumoral microglia, TAMCs and T-cell infiltration. These inflammatory changes may prolong survival of CT-2A glioma-bearing mice.

TMIC-58. LEVERAGING VIRO-IMMUNOTHERAPY BY TARGETING IGF2-IGF1R SIGNALING

Abstract While an FDA-approved oncolytic herpes simplex-1 virus (oHSV) has shown therapeutic promise with superior safety, accumulating clinical data revealed that its therapeutic efficacy is observed in a small subset of patients. Here, we show that NFκB-mediated Insulin-like Growth Factor 2 (IGF2)/Insulin-like Growth Factor-1 Receptor (IGF1R) signaling pathway as a key modulator for oHSV therapy-mediated tumor resistance. RNA sequencing on the oHSV-infected primary glioblastoma (GBM) and breast cancer (BC) cells identified IGF2 as one of the top 10 upregulated secretomes. Transcriptomic analysis also revealed significant upregulation/enrichment of genes related to IGF2-IGF1R-MAPK pathway in oHSV-treated tumor cells. Infection with oHSV in GBM and BC cells up-regulated IGF2 transcription via direct binding of NFκB in the IGF2 promoter 3. Compromising the IGF2-IGF1R signaling pathway using IGF2 neutralizing antibody significantly increased oHSV-mediated tumor cell killing in vitro and mice survival in vivo. To circumvent this and promptly translate our findings in clinical settings, we have developed a novel oHSV, oHSV-D11mt, by incorporating modified IGF2R domain 11 into the parental oHSV genome to function as IGF2 decoy receptor as a single-agent modality. oHSV-D11mt specifically bind to IGF2 not IGF1, blocks oHSV-induced IGF2-IGF1R signaling, increases tumor cell killing, decreases oHSV-induced neutrophils/PMN-MDSCs infiltration, immune suppressive/proangiogenic cytokine secretion, and increases CD8+ cytotoxic T lymphocytes (CTLs) activation, resulting in increased virus propagation and mice survival. These findings suggest that IGF2-IGF1R signaling is a novel target to overcome oHSV therapy resistance and oHSV-D11mt could be used for improved viro-immunotherapy.

EXTH-79. BREAKING THE BARRIER: ADOPTIVE CELLULAR THERAPY HALTS GLIOMA IMMUNOSUPPRESSION BY TARGETING MYELOID-DERIVED SUPPRESSOR CELL MIGRATION AND CELL CYCLE MECHANISMS

Abstract INTRODUCTION Our group previously demonstrated that adoptive cellular therapy (ACT) significantly increased overall survival in mice across several brain cancer models. ACT remodels the tumor microenvironment (TME) in a pleiotropic manner, and specifically depletes immunosuppressive cells like myeloid-derived suppressor cells (MDSCs) which are a significant barrier to efficacious immunotherapy responses. This led us to hypothesize that ACT depletes MDSCs from the glioma microenvironment by inhibiting MDSC proliferation and migration, thereby overcoming glioma immunosuppression. METHODS We quantified MDSC cell cycle, apoptosis, and localization in the TME and peripheral lymphoid tissues using flow cytometry as well as chemokines in the TME from glioma-bearing mice treated with ACT. C57BL/6J mice intracranially received KR158b-luciferase glioma and were treated with ACT. After treatment, MDSC cell cycle was quantified using bromodeoxyuridine and 7-aminoactinomycin to define cell cycle phases. MDSC apoptosis and cell death states were quantified by measuring annexin-V and propidium iodide. Anatomic localization of MDSCs was measured by comparing host (CD45.2) and transferred HSC-derived cell markers (CD45.1) in the TME and spleen. A multiplex array was performed using tumor lysates to quantify cytokine and chemokine levels within the TME. RESULTS We observed that MDSCs in the TME were significantly less proliferative and exhibited significantly increased levels of apoptosis and cell death after ACT. HSC-derived MDSCs were significantly enriched in the spleen of ACT-treated mice, but both transferred HSC-derived MDSCs and host MDSCs were depleted in the TME. Finally, ACT significantly decreased several chemokines in the TME, including interleukin-16 and monocyte chemoattractant protein-5. CONCLUSION These results indicate that ACT shifts MDSC cell cycle states away from proliferation towards cell death, and inhibits peripheral recruitment to the TME. Our investigation revealed novel, myeloproliferative chemokines that are decreased by ACT, representing a novel mechanism underlying disrupted MDSC migration to the glioma microenvironment and ultimately to overcoming glioma-mediated immunosuppression.

ANGI-03. GLIOMA STEM CELLS INDUCE VASECTASIA – A NON-ANGIOGENIC BRAIN TUMOUR NEOVASCULARIZATION PROCESS DRIVEN BY INTERCELLULAR EGFR TRANSFER THROUGH EXTRACELLULAR VESICLES

Abstract Tumour neovascularization in glioblastoma (GBM) is abundant, abnormal and poorly understood, and it likely involves multiple co-existing mechnanisms with distinct modes of regulation. Since glioma stem cells (GSCs) engage in bidirectional interactions with the vasculature we dissected the resulting vascular responses elicited by either proneural (PN), or mesenchymal (MES) GSC subtypes established from patients isolates and able to drive progression of orthotopic intracranial xenografts in mice. We report here that while PN-GSCs elicited mostly angiogenesis-type vascular capillary growth processes, their MES-GSC counterparts elicited non-angiogenic growth of enlarged vessels (vasectesia), paradoxically associated with reduced blood vessel density. Vasectasia was dependent on the expression of oncogenic EGFR/EGFRvIII by MES-GSCs, and on the related kinase/signalling activity, as EGFR/EGFRvIII gene disruption and Dacomitinib treatment suppressed formation of large blood vessels in vivo and resulted in a preponderance of a small calliber (angiogenic) vasculature throughout xenografts. Notably, the analysis of spatial transcriptomes and single cell RNA sequencing revealed distinct molecular traits of endothelial cells asociated with vasectasia and angiogesis, the former enriched in Birc5, but depleted for angiogenic tip cell markers (Vegfr2, Apln). Mechnaistically, vasectasia was linked to the intercellular transfer of EGFR from MES-GSCs to endothelial cells resulting in ectopic activation of EGFR signalling in the endothelial cell bacground, both in vitro and in a model of in vivo blood vessel recruitment into implanted matrigel pellets. Simultaneous pharmacological blockade of both, angiogenesis (anti-VEGR2) and vasectasia (Dacomitinib) lead to a marked increase in survival of mice with aggressive GBM lesions initiated by MES-GSCs. We suggest that, vasectasia driven by intercellular transfer of oncogenic EGFR may represent a new, subtype-specific mechanism of GBM neovascularization, and a plausible target for agents aiming to modify vascular tumour microenvironment.

Exposure to live saprophytic Leptospira before challenge with a pathogenic serovar prevents severe leptospirosis and promotes kidney homeostasis

Previous studies demonstrated that Leptospira biflexa, a saprophytic species, triggers innate immune responses in the host during early infection. This raised the question of whether these responses could suppress a subsequent challenge with pathogenic Leptospira. We inoculated C3H/HeJ mice with a single or a double dose of L. biflexa before challenge with a pathogenic serovar, Leptospira interrogans serovar Copenhageni FioCruz (LIC). Pre-challenge exposure to L. biflexa did not prevent LIC dissemination and colonization of the kidney. However, it rescued weight loss and mouse survival thereby mitigating disease severity. Unexpectedly, there was correlation between rescue of overall health (weight gain, higher survival, lower kidney fibrosis marker ColA1) and higher shedding of LIC in urine. This stood in contrast to the L. biflexa unexposed LIC challenged control. Immune responses were dominated by increased frequency of effector T helper (CD4+) cells in spleen, as well as significant increases in serologic IgG2a. Our findings suggest that exposure to live saprophytic Leptospira primes the host to develop Th1 biased immune responses that prevent severe disease induced by a subsequent challenge with a pathogenic species. Thus, mice exposed to live saprophytic Leptospira before facing a pathogenic serovar may withstand infection with far better outcomes. Furthermore, a status of homeostasis may have been reached after kidney colonization that helps LIC complete its enzootic cycle.

Exposure to live saprophytic Leptospira before challenge with a pathogenic serovar prevents severe leptospirosis and promotes kidney homeostasis.

Previous studies demonstrated that Leptospira biflexa, a saprophytic species, triggers innate immune responses in the host during early infection. This raised the question of whether these responses could suppress a subsequent challenge with pathogenic Leptospira. We inoculated C3H/HeJ mice with a single or a double dose of L. biflexa before challenge with a pathogenic serovar, Leptospira interrogans serovar Copenhageni FioCruz (LIC). Pre-challenge exposure to L. biflexa did not prevent LIC dissemination and colonization of the kidney. However, it rescued weight loss and mouse survival thereby mitigating disease severity. Unexpectedly, there was correlation between rescue of overall health (weight gain, higher survival, lower kidney fibrosis marker ColA1) and higher shedding of LIC in urine. This stood in contrast to the L. biflexa unexposed LIC challenged control. Immune responses were dominated by increased frequency of effector T helper (CD4+) cells in spleen, as well as significant increases in serologic IgG2a. Our findings suggest that exposure to live saprophytic Leptospira primes the host to develop Th1 biased immune responses that prevent severe disease induced by a subsequent challenge with a pathogenic species. Thus, mice exposed to live saprophytic Leptospira before facing a pathogenic serovar may withstand infection with far better outcomes. Furthermore, a status of homeostasis may have been reached after kidney colonization that helps LIC complete its enzootic cycle.