Days Of Tumor Growth Research Articles

TMIC-22. MYELOID CELLS INDUCE IMMUNOSUPPRESSION AND HYPORESPONSIVENESS OF CHIMERIC ANTIGEN RECEPTOR T CELLS IN THE NEURONAL MICROENVIRONMENT OF GLIOBLASTOMA

Abstract Chimeric antigen receptor T (CART)-cell therapy has shown little success in glioblastoma and other solid tumors, unlike in hematologic malignancies. The immunosuppressive milieu and intratumoral heterogeneity are some obstacles limiting the effectiveness of CART-cell therapy in glioblastoma. Our goal in this study is to investigate how CART-cell transcriptional adaptations and cellular interactions in the glioblastoma microenvironment drive T-cell hyporesponsiveness. Using a human neocortical slice model, we performed physically interacting cell sequencing (PIC-seq) to investigate the transcriptional diversity of NKG2D CAR-T cells and control T-cells in the glioblastoma ecosystem. Through the integration of spatially resolved T-cell receptors sequencing and PIC-seq, we investigated the cellular and receptor-ligand interactions in CART-treated glioblastoma cells. Gene regulatory networks were reconstructed to identify key transcriptional regulators of CD8 CART-cell dysfunction. We used in-silico perturbation to understand transcriptional drivers that could potentially enhance the cytotoxic response of CAR-Ts. Primary patient-derived glioma cells were inoculated into human neocortical slices from surgical access tissue specimens. After 3 days of tumor growth, slices were treated with NKG2D CARTs or control T-cells. We observed that after 2 days of anti-tumor T-cell response in the CART group, tumor growth did not differ between conditions. After 7 days of treatment, PIC-seq was used to profile CARTs (tomato+) and tumor cells (GFP+) as well as physically connected cells (+/+). We identified a transcriptional shift towards T-cell exhaustion in CART cells compared to T-cells lacking the NKG2D construct. This transcriptional phenotype was driven by myeloid-CART and myeloid-tumor interaction. We identified that tumor-associated macrophages with enhanced phagocytic function in proximity to mesenchymal-like tumor cells located within hypoxic niches are the main drivers of T-cell dysfunction. Gene regulatory network analysis demonstrated that MAF and BACH2 are key transcriptional regulators of CART-cell function. Myeloid-tumor and myeloid-T-cell interactions promote an immunosuppressive microenvironment and hyporesponsive T-cells in glioblastoma. Our data has the potential to catalyze the re-engineering and optimization of CART-cell therapies with sustained cytotoxic effect against glioblastoma.

Nanomedicine Targeting Cuproplasia in Cancer: Labile Copper Sequestration Using Polydopamine Particles Blocks Tumor Growth In Vivo through Altering Metabolism and Redox Homeostasis.

Copper plays critical roles as a metal active site cofactor and metalloallosteric signal for enzymes involved in cell proliferation and metabolism, making it an attractive target for cancer therapy. In this study, we investigated the efficacy of polydopamine nanoparticles (PDA NPs), classically applied for metal removal from water, as a therapeutic strategy for depleting intracellular labile copper pools in triple-negative breast cancer models through the metal-chelating groups present on the PDA surface. By using the activity-based sensing probe FCP-1, we could track the PDA-induced labile copper depletion while leaving total copper levels unchanged and link it to the selective MDA-MB-231 cell death. Further mechanistic investigations revealed that PDA NPs increased reactive oxygen species (ROS) levels, potentially through the inactivation of superoxide dismutase 1 (SOD1), a copper-dependent antioxidant enzyme. Additionally, PDA NPs were found to interact with the mitochondrial membrane, resulting in an increase in the mitochondrial membrane potential, which may contribute to enhanced ROS production. We employed an in vivo tumor model to validate the therapeutic efficacy of PDA NPs. Remarkably, in the absence of any additional treatment, the presence of PDA NPs alone led to a significant reduction in tumor volume by a factor of 1.66 after 22 days of tumor growth. Our findings highlight the potential of PDA NPs as a promising therapeutic approach for selectively targeting cancer by modulating copper levels and inducing oxidative stress, leading to tumor growth inhibition as shown in these triple-negative breast cancer models.

Nano-Pulse Stimulation Treatment Inhibits Pan02 Murine Pancreatic Tumor Growth and Induces a Long-Term Adaptive Immune Response with Abscopal Effects When Combined with Immune-Enhancing Agents.

Pancreatic cancer is associated with a poor prognosis and immunotherapy alone has not demonstrated sufficient efficacy in the treatment of nonresectable tumors. Nano-Pulse Stimulation™ therapy (NPS™) applies nanosecond electric pulses that lead to regulated cell death, exposing tumor antigen to the immune system. To establish a primary Pan02 tumor, mice were intradermally injected with Pan02 cells into the right flank. Secondary, rechallenge tumors and distal, secondary tumors (abscopal response) were established by injecting Pan02 cells into the opposite left flank. After 5 days of tumor growth, one of the tumors was treated with NPS, followed by injection with an immune-enhancing agent to stimulate an immune response. Growth of the treated primary tumor and untreated rechallenge tumors (injected 60-days post-treatment) or distal secondary tumors (injected simultaneously with the primary) was monitored. NPS in combination with the adjuvant and TLR agonist, resiquimod (RES), was the optimal treatment regimen for both eliminating a primary Pan02 tumor as well as inhibiting growth of a Pan02 cell rechallenge tumor. This inhibition of the rechallenge tumor injected 2 months after eliminating the primary tumor suggests a long-term immune response had been stimulated. Additional support for this came from the observations that depleting CD8+ T-cells reduced inhibition of rechallenge tumor growth by 35% and rechallenge tumors had 3-fold more CD8+ T-cells than tumors injected after surgical resection of the primary tumor. When the NPS-treated tumor was immediately injected with the anti-OX40 antibody to agonize the function of the costimulatory T cell receptor, OX40, up to 80% of untreated abscopal tumors were eliminated. NPS plus RES was the most effective at both eliminating a primary tumor and inhibiting a rechallenge tumor. NPS treatment followed by injection of aOX40 was the most effective at inhibiting the growth of an untreated abscopal tumor.

Rapid non-invasive tracking of splenomegaly with hands-free 3D ultrasound for on-demand assessment of immune disorders and cancer

Abstract Splenomegaly is an important measure of cancer progression and immune disorders. Magnetic resonance imaging (MRI) and computed tomography (CT) are the standard in the field but are not optimal for preclinical studies due to their high cost, low throughput and reliance on contrast agents for soft tissue. Here, we report the use of a novel 3D automated ultrasound scanner (Vega, Revvity Inc.) for the rapid assessment of spleen volume in two murine models (cancer and lupus). Cancer cohort: Tumor-bearing (4T1 breast cancer, right flank) mice were imaged alongside control mice at five timepoints over 25 days. Spleen volume was found to increase 18-fold (30±3 mm3 to 545±105 mm3) after 25 days of tumor growth (938±77 mm3). Lupus cohort: MRL/lpr mice were allowed to age until they developed lymphoproliferation (12-14 weeks) and then imaged biweekly until 20 weeks of age. A 300-fold increase in calculated spleen volume by 18 weeks of age in MRL/ lpr mice was observed. Kidney volume doubled and a statistical increase (p<0.05) in vascular density was observed (using acoustic angiography), indicating systemic disease progression. Average acquisition times for both cohorts were 30 seconds per mouse, showing excellent applicability of spleen volume as a measurement of disease progression compared to more laborious techniques (e.g. MRI). Future work in this area will explore the application of machine learning and AI-assisted auto segmentation tools for higher throughput data analysis.

Abstract 6516: Single-cell protein activity inference reveals lymphocytic immune infiltration in diffuse intrinsic pontine glioma induced by focused ultrasound

Abstract Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor, located in the brainstem, and traditionally thought to be immune-excluded with no significant infiltration by cytotoxic T-cells, and therefore not amenable to immunotherapies. Focused ultrasound technology (FUS) for drug delivery is being investigated for DIPG in both preclinical and clinical trial settings. In this study, we sought to study the effects of FUS and radiation therapy (RT) on immune cell infiltration in DIPG tumors to assess their ability to alter the immune microenvironment and potentially enhance combination immunotherapies. Specifically, we generated single-cell RNAseq profiles for 27,780 cells from syngeneic orthotopic DIPG tumors (KPAP cell line containing H3K27M mutation, ATRX and P53 loss, and PDGFRA overexpression) after 14 days of tumor growth across the following treatment arms: vehicle control, FUS, and RT (n=2 each group). FUS was applied by 4-point sonication around the tumor with a pressure of 0.6 Mpa at days 8 and 11 post tumor implantation (PTI). RT was administered in fractions of 3 Gy daily from days 8 to 11 PTI (12Gy total). We leveraged a set of algorithms for network-based inference of regulatory protein activity to infer a set of context-matched gene regulatory networks from which protein activity can be inferred by expression of downstream targets. By this approach, we identified distinct clusters of immune cells segregating by cell type as inferred by singleR and activity of known lineage markers. Strikingly, we found that both RT and FUS significantly depleted a TGFb-expressing population of immune-suppressive macrophages as compared to vehicle controls. Importantly, FUS only additionally increased T-cell infiltration into the tumor micro-environment over three-fold as compared to vehicle controls, which was not observed in response to RT. Further protein validation is underway. This has significant implications for therapeutic combinations of FUS-mediated drug delivery with immune checkpoint inhibitors in DIPG, such that in addition to drug delivery, FUS-mediated immune modulatory effects may provide more proinflammatory response to enhance immunotherapy. Citation Format: Erin White, Hong-Jian Wei, Ester Calvo Fernández, Hanna Minns, Jovana Pavisic, Robyn Gartrell, Cheng-Chia Wu, Andrea Califano, Aleksandar Obradovic. Single-cell protein activity inference reveals lymphocytic immune infiltration in diffuse intrinsic pontine glioma induced by focused ultrasound [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6516.

Dynamics of cachexia-associated inflammatory changes in the brain accompanying intra-abdominal fibrosarcoma growth in Wistar rats

Accumulated data indicate that inflammation affecting brain structures participates in the development of cancer-related cachexia. However, the mechanisms responsible for the induction and progression of cancer-related neuroinflammation are still not fully understood. Therefore, we studied the time-course of neuroinflammation in selected brain structures and cachexia development in tumor-bearing rats. After tumor cells inoculation, specifically on the 7th, 14th, 21st, and 28th day of tumor growth, we assessed the presence of cancer-associated cachexia in rats. Changes in gene expression of inflammatory factors were studied in selected regions of the hypothalamus, brain stem, and circumventricular organs. We showed that the initial stages of cancer growth (7th and 14th day after tumor cells inoculation), are not associated with cachexia, or increased expression of inflammatory molecules in the brain. Even when we did not detect cachexia in tumor-bearing rats by the 21st day of the experiment, the inflammatory brain reaction had already started, as we found elevated levels of interleukin 1 beta, interleukin 6, tumor necrosis factor alpha, and glial fibrillary acidic protein mRNA levels in the nucleus of the solitary tract. Furthermore, we found increased interleukin 1 beta expression in the locus coeruleus and higher allograft inflammatory factor 1 expression in the vascular organ of lamina terminalis. Ultimately, the most pronounced manifestations of tumor growth were present on the 28th day post-inoculation of tumor cells. In these animals, we detected cancer-related cachexia and significant increases in interleukin 1 beta expression in all brain areas studied. We also observed significantly decreased expression of the glial cell activation markers allograft inflammatory factor 1 and glial fibrillary acidic protein in most brain areas of cachectic rats. In addition, we showed increased expression of cluster of differentiation 163 and cyclooxygenase 2 mRNA in the hypothalamic paraventricular nucleus, A1/C1 neurons, and area postrema of cachectic rats. Our data indicate that cancer-related cachexia is associated with complex neuroinflammatory changes in the brain. These changes can be found in both hypothalamic as well as extrahypothalamic structures, while their extent and character depend on the stage of tumor growth.

Antitumor effect of recombinant interferon-gamma in an experimental model of Ehrlich’s bilateral solid carcinoma

Introduction.Immunotherapy, which is part of the complex and combined cancer therapy, is one of the priority areas in the treatment of cancer patients. However, the effectiveness of the use of immunotherapeutic drugs of the latest generation is not so high, and in some patients the effect of therapy was short-lived. Factors that prevent the full realization of the antitumor effect of cytostatics and immunopreparations may be the features of the antigenic composition of the tumor, as well as its cellular and stromal microenvironment. These facts contributed to the development of a new strategy, designated as immunoredaction of cancer by exposure to various biologically active agents that can change the body – tumor ratio in favor of the patient and make the tumor available for the implementation of antitumor effects of the host immune system.The study objective– experimental substantiation of the development of new immunotherapeutic approaches in the treatment of aggressive forms of cancer.Materials and methods.An experimental study of the effect of human recombinant interferon-gamma (IFNγ) on the growth of Ehrlich’s carcinoma during subcutaneous bilateral transplantation of tumor cells to animals was carried out. Transplantation of Ehrlich’s carcinoma to male F1 hybrids (SWAhC57Bl6) was performed by subcutaneous injection of 2.0 × 106 tumor cells (7‑day culture) in 0.1 ml of suspension into the lateral surface of the right and left femur with imitation of multicentric growth.Results.A day after the course of drug administration (day 6 of tumor node growth), the effect of suppressing tumor growth in relation to growth in the control group was noted. The maximum inhibition effect of 19.8 % (p<0.05) of tumor growth was obtained 5 days after the course of the drug (10 days of tumor growth, right node) and 18.5 % (p<0.001) 9 days after administration (14 days of tumor growth, left node).Conclusion.Thus, a distinct, statistically significant antitumor effect of IFNγ was established in relation to a tumor with a multicentric growth pattern.

Mitochondrial therapy can inhibit melanoma development.

e21571 Background: Mitochondria of tumor cells undergo adaptive changes for even more active reproduction of tumor cells in the acidic and hypoxic microenvironment of tumor tissue. The purpose of this study was to evaluate antitumor effect of mitochondrial therapy in BALB/c Nude mice of both genders with B16/F10 melanoma growth. Methods: Male (n = 10) and female (n = 10) BALB/c Nude mice were injected subcutaneously with 0.5 ml of a suspension of B16/F10 mouse melanoma tumor cells in saline at a dilution of 1:20. Mitochondria (MC) were isolated from rat liver. 24 hours after subcutaneous transplantation of B16/F10 melanoma, mice were intraperitoneally injected with freshly isolated mitochondria (3.3 mg of protein per animal in 0.3 ml of saline). Further, mitochondrial therapy (MC therapy) was carried out according to the scheme on days 3, 5, 9, 13, 16, 19, 21. Male BALB/c Nude mice with subcutaneous inoculation of B16/F10 melanoma receiving intraperitoneal injections with 0.3 ml of saline served as controls. Results: Subcutaneous tumors in mice of both genders could be determined on the 5th day from the moment of tumor transplantation. In males, the regressive effect of MC therapy was recorded from the 8th day of tumor growth. The average tumor volume in males with MC therapy on day 8 was 3.0 times less than in the control group. On days 12 and 15, the regressive difference in tumor volumes between the groups was 1.8 times (p < 0.05) (MC therapy). On day 19, a slower tumor growth was recorded in the group with MC therapy, where the tumor volume was 2 times lower compared to the control volumes. At the end of the experiment on day 22, the difference in the average tumor volumes was 3.2 times, i.e. a significant inhibition of tumor growth was determined in males treated with MC therapy. A sizeable tumor node in females treated with MC therapy formed longer than in males, remaining as a flat tumor spot for up to 8 days. The inhibition of the growth of a large tumor node was determined on day 12 of melanoma growth, and the difference with the control values was 1.8 times (p < 0.05). On day 15, the tumor volumes in females with MC therapy decreased by 2.2 times compared with the control values, on day 19 - by 2.1 times. As a result, by the end of the experiment (day 22), the difference with the control group was 2.7 times. Conclusions: MC therapy inhibits the growth of B16/F10 melanoma in male and female BALB/c Nude mice. Formation of a sizeable tumor node and the start the tumor growth inhibition differs in time depending on the gender of experimental animals.

TAMI-64. IMMUNE ENVIRONMENT SHAPES GLIOBLASTOMA HETEROGENEITY IN TIME AND SPACE

Abstract Glioblastomas are embedded into an immunosuppressive microenvironment resulting in limited success of immunotherapies. Although, we and others observed defined myeloid-tumor crosslinks reducing T cell homing and activation, the spatial context of these interactions remained unexplored. Here, we provide evidence, that local T cell infiltration results in a defined activation of myeloid cells causing transcriptional reprogramming of tumor cells reminiscent of reactive transformation in mature astrocytes. Through integration of spatially resolved transcriptomics and imaging mass cytometry (n=18, 39 protein glioma panel) we mapped defined transcriptional responses in areas of high or low T cell infiltration respectively. Functional analysis revealed that areas of large T cell infiltration are enriched for glial (CHI3L1, GFAP and VIM) and inflammatory genes (HLA-DRA, C3, CCL4, CCL3). We found that marker genes of common reactive states in mature astrocytes significantly overlap with the reactive immune program of glioblastoma cells (f-score 0.76, p=2.2e-10). Increased numbers of CD163+ cells were found in surrounding areas of T cell infiltration and spatially linked to defined immunosuppressive release of IL10, recently reported as a major driver of T cell exhaustion. To support our findings, we injected a primary glioblastoma cell line into cortex slices of three different human and rodent donors. After 7 days of tumor growth, we performed scRNA-sequencing of FACS-sorted tumor cells and baseline cell culture cells. Compared to baseline, we found cells of all reported transcriptional states, confirming the dynamic adaptation of cells within a neural environment. In elderly donors, a significant accumulation of reactive immune programs (ANOVA p< 0.001) was observed. Immunostainings confirmed an increased myeloid cell activation in these donors. Our data suggest that inflammatory stimuli in the glioblastoma microenvironment cause transcriptional reprogramming in glioblastoma similar to inflammatory transformation of reactive astrocytes. The spatial exclusivity of these programs highlights the value of a spatial perspective on heterogeneity.

Establishment and Validation of CyberKnife Irradiation in a Syngeneic Glioblastoma Mouse Model.

Simple SummaryStereotactic radiosurgery (SRS) provides precise high-dose irradiation of intracranial tumors. However, its radiobiological mechanisms are not fully understood. This study aims to establish CyberKnife SRS on an intracranial glioblastoma tumor mouse model and assesses the early radiobiological effects of radiosurgery. Following exposure to a single dose of 20 Gy, the tumor volume was evaluated using MRI scans, whereas cellular proliferation and apoptosis, tumor vasculature, and immune response were evaluated using immunofluorescence staining. The mean tumor volume was significantly reduced by approximately 75% after SRS. The precision of irradiation was verified by the detection of DNA damage consistent with the planned dose distribution. Our study provides a suitable mouse model for reproducible and effective irradiation and further investigation of radiobiological effects and combination therapies of intracranial tumors using CyberKnife.CyberKnife stereotactic radiosurgery (CK-SRS) precisely delivers radiation to intracranial tumors. However, the underlying radiobiological mechanisms at high single doses are not yet fully understood. Here, we established and evaluated the early radiobiological effects of CK-SRS treatment at a single dose of 20 Gy after 15 days of tumor growth in a syngeneic glioblastoma-mouse model. Exact positioning was ensured using a custom-made, non-invasive, and trackable frame. One superimposed target volume for the CK-SRS planning was created from the fused tumor volumes obtained from MRIs prior to irradiation. Dose calculation and delivery were planned using a single-reference CT scan. Six days after irradiation, tumor volumes were measured using MRI scans, and radiobiological effects were assessed using immunofluorescence staining. We found that CK-SRS treatment reduced tumor volume by approximately 75%, impaired cell proliferation, diminished tumor vasculature, and increased immune response. The accuracy of the delivered dose was demonstrated by staining of DNA double-strand breaks in accordance with the planned dose distribution. Overall, we confirmed that our proposed setup enables the precise irradiation of intracranial tumors in mice using only one reference CT and superimposed MRI volumes. Thus, our proposed mouse model for reproducible CK-SRS can be used to investigate radiobiological effects and develop novel therapeutic approaches.

Effect of 5-hydroxypyrimidine derivatives on tumor growth and cytokine concentration in blood serum of female CBA mice with cervical cancer (RSHM-5)

The effects of intraperitoneal administration of SNK-411 (2-isobutyl-4,6-dimethyl-5-hydroxypyrimidine) in a dose of 25 mg/kg (the total dose of 350 mg/kg) and SNK-578 (hydrochloride of 2-isobutyl-4,6-dimethyl-5- hydroxypyrimidine) in a dose of 10 mg/kg (the total dose of 140 mg/kg) on tumor growth and concentration of cytokines in the blood serum were studied in female CBA mice. Substances were administrated from the 2nd to 15th days of tumor development. Tumor growth inhibition (TGI) and serum cytokine level were studied on the 7th day after the end of compounds administration (21st day of tumor growth). In intact control group (n=10) median tumor mass was 1255 mg. TGI in the group of animals treated with SNK-411 was 47%; in the group of mice treated with SNK-578 TGI was 87%, tumor mass demonstrated 7.4-fold reduction. Serum concentrations of cytokines (IL-6, IL-10, IL-17A and IFN-γ) in tumor-bearing group of mice were higher versus the intact control group by 229%, 40%, 60% and 81%, respectively. Highly active SNK-578 decreased concentrations of prooncogenic IL-10, IL-17A and proinflammatory IL-6, by 61%, 70% and 29% as compared to tumor-bearing control group. SNK-411 decreased concentrations of prooncogenic IL-10 and IL-17A by 48% and 60%, respectively, and did not affect concentration of IL-6. Taking into consideration that IL-6 participates in autoimmune reactions, we can assume that the immune control is one of the crucial mechanisms of antitumor effect of SNK-578. All results are statistically significant.

Abstract 6128: Evaluation of therapeutic efficacy of combination of 4HPR and SAHA for inhibition of growth of human glioblastoma T98G xenografts in zebrafish

Abstract Glioblastoma is the most aggressive and deadly primary brain cancer in humans. Despite the current treatments using surgery, radiotherapy, and chemotherapy, glioblastoma reappears due to drug resistance. New approaches for treatment of glioblastoma suggest that combination of two drugs may overcome the drug resistance. In this study, we have evaluated the efficacy of the combination of N-(4-hydroxphenyl) retinamide (4HPR) and suberoylanilide hydroxamic acid (SAHA) for inhibition of human glioblastoma T98G cells in a zebrafish xenograft model in our laboratory. Both 4HPR and SAHA have been approved by the United States Food and Drug Administration for treatment of cancers in the clinics. The synthetic retinoid 4HPR works via retinoic acid receptor signaling pathways to inhibit cancer cell proliferation and induce apoptosis, while SAHA is a histone deacetylase (HDAC) inhibitor that targets class I, II, and IV HDACs for controlling growth of cancer cells. Although rodent models have provided valuable insights into the biology and pathology of glioblastoma, these models are frankly unsuitable and increasingly unaffordable for high-throughput screening and profiling of novel therapeutic agents for rapid translation to the clinics. The optical transparency of zebrafish embryos and larvae provides the unique opportunity to track tumors in a way that is impossible in rodent models. Small size of zebrafish embryos allows for study design in small plates, making them a more useful laboratory system than other cancer models. Zebrafish lack a fully functioning adaptive immune response until 4 days post-fertilization, allowing rejection-free xenografts of human tumors during this period. Recently, zebrafish xenograft models have emerged as a new powerful tool for studying different types of human tumors including glioblastoma and for evaluating therapeutic efficacy of different drugs alone and in combination. Organ systems and cell types are common in human and zebrafish. Tumors that occur in the same organs in human and zebrafish look and behave alike, often share common genetic makeups, and employ basic mechanisms for tumor formation, expansion, and spread. Zebrafish xenograft model of human glioblastoma offers us a major opportunity for discovering key pathways of this tumor growth and for evaluating efficacy of a novel combination therapy. We loaded exponentially grown T98G cells labeled with CellTrace™ Red into the glass needle (0.75 mm internal diameter without filament, World Precision Instruments, FL) at a density of about 1 × 106 cells/200 µl. Using a micro-injector, each anesthetized zebrafish larva was microinjected with about 100 T98G cells into the duct of Cuvier at 1.5 - 3.0 psi. After injection, larvae were transferred to fresh egg water for recovery during 1 h at 28 ± 0.5°C and then incubated at 33°C for the rest of the experiment. After 2 days of tumor growth, we treated zebrafish larvae with 4 µM 4HPR and 8 µM SAHA alone and in combination and incubated for 3 days. Results showed that 4HPR and SAHA alone inhibited growth of T98G xenografts in zebrafish, but the combination of the two drugs most significantly reduced T98G cell proliferation inside of the zebrafish. Our work suggested that the zebrafish xenograft model could be useful to identify the therapeutic efficacy of combination of drugs in the treatment of glioblastoma. Citation Format: Firas Khathayer, Katie L. Kathrein, Mitzi Nagarkatti, Swapan K. Ray. Evaluation of therapeutic efficacy of combination of 4HPR and SAHA for inhibition of growth of human glioblastoma T98G xenografts in zebrafish [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6128.

Pivotal role of NF-κB in cellular senescence of experimental pituitary tumours.

The molecular mechanisms underlying the capability of pituitary tumours to avoid unregulated cell proliferation are still not well understood. However, the NF-κB transcription factor, which is able to modulate not only cellular senescence but also tumour progression, has emerged as a targeted candidate. This work was focused on the NF-κB role in cellular senescence during the progression of experimental pituitary tumours. Also, the contribution of the signalling pathways in senescence-associated NF-κB activation and the senescence-associated secretory phenotype (SASP) and pro-survival-NF-κB target genes transcription were analysed. A robust NF-κB activation was seen at E20-E40 of tumour development accompanied by a marked SA-β-Gal co-reactivity in the tumour pituitary parenchyma. The induction of TNFα and IL1-β as specific SASP-related NF-κB target genes as well as Bcl-2 and Bcl-xl pro-survival genes was shown to be accompanied by increases in the p-p38 MAPK protein levels, starting at the E20 stage and strengthening from 40 to 60 days of tumour growth. It is noteworthy that p-JNK displayed a similar pattern of activation during pituitary tumour development, while p-AKT and p-ERK1/2 were downregulated. By employing a pharmacological strategy to abrogate NF-κB activity, we demonstrated a marked reduction in SA-β-Gal activity and a slight decrease in Ki67 immunopositive cells after NF-κB blockade. These results suggest a central role for NF-κB in the regulation of the cellular senescence programme, leading to the strikingly benign intrinsic nature of pituitary adenomas.

The CXCR2/CXCL2 signalling pathway – An alternative therapeutic approach in high-grade glioma

ObjectiveBesides VEGF, alternative signalling via CXCR2 and its ligands CXCL2/CXCL8 is a crucial part of angiogenesis in glioblastoma. Our aim was to understand the role of CXCR2 for glioma biology and elucidate the therapeutic potential of its specific inhibition. MethodsGL261 glioma cells were implanted intracranially in syngeneic mice. The 14 or 7 days of local or systemic treatment with CXCR2-antagonist (SB225002) was initiated early on the day of tumour cell implantation or delayed after 14 days of tumour growth. Glioma volume was verified using MRI before and after treatment. Immunofluorescence staining was used to investigate tumour progression, angiogenesis and microglial behaviour. Furthermore, in vitro assays and gene expression analyses of glioma and endothelial cells were performed to validate inhibitor activity. ResultsCXCR2-blocking led to significantly reduced glioma volumes of around 50% after early and delayed local treatments. The treated tumours were comparable with controls regarding invasiveness, proliferation and apoptotic cell activity. Furthermore, no differences in CXCR2/CXCL2 expression were observed. However, immunostaining revealed reduction in vessel density and accumulation of microglia/macrophages, whereas interaction of these myeloid cells with tumour vessels was enhanced. In vitro analyses of the CXCR2-antagonist showed its direct impact on proliferation of glioma and endothelial cells if used at higher concentrations. In addition, expression of CXCR2/CXCL2 signalling genes was increased in both cell types by SB225002, but VEGF-relevant genes were unaffected. ConclusionThe CXCR2-antagonist inhibited glioma growth during tumour initiation and progression, whereas treatment was well-tolerated by the recipients. Thus, the CXCR2/CXCL2 signalling represents a promising therapeutic target in glioma.

Erratum to: The anti-tumor effect of the quinoline-3-carboxamide tasquinimod: blockade of recruitment of CD11b+ Ly6Chi cells to tumor tissue reduces tumor growth.

Previous work has demonstrated immunomodulatory, anti-tumor, anti-metastatic and anti-angiogenic effects of the small molecule quinoline-3-carboxamide tasquinimod in pre-clinical cancer models. To better understand the anti-tumor effects of tasquinimod in transplantable tumor models, we have evaluated the impact of the compound both on recruitment of myeloid cells to tumor tissue and on tumor-induced myeloid cell expansion as these cells are known to promote tumor development. Mice bearing subcutaneous 4 T1 mammary carcinoma tumors were treated with tasquinimod in the drinking water. A BrdU-based flow cytometry assay was utilized to assess the impact of short-term tasquinimod treatment on myeloid cell recruitment to tumors. Additionally, long-term treatment was performed to study the anti-tumor effect of tasquinimod as well as its effects on splenic myeloid cells and their progenitors. Myeloid cell populations were also immune-depleted by in vivo antibody treatment. Short-term tasquinimod treatment did not influence the proliferation of splenic Ly6Chi and Ly6Ghi cells, but instead reduced the influx of Ly6Chi cells to the tumor. Treatment with tasquinimod for various periods of time after tumor inoculation revealed that the anti-tumor effect of this compound mainly operated during the first few days of tumor growth. Similar to tasquinimod treatment, antibody-mediated depletion of Ly6Chi cells within that same time frame, caused reduced tumor growth, thereby confirming a significant role for these cells in tumor development. Additionally, long-term tasquinimod treatment reduced the splenomegaly and expansion of splenic myeloid cells during a later phase of tumor development. In this phase, tasquinimod normalized the tumor-induced alterations in myeloerythroid progenitor cells in the spleen but had only limited impact on the same populations in the bone marrow. Our results indicate that tasquinimod treatment reduces tumor growth by operating early after tumor inoculation and that this effect is at least partially caused by reduced recruitment of Ly6Chi cells to tumor tissue. Long-term treatment also reduces the number of splenic myeloid cells and myeloerythroid progenitors, but these effects did not influence established rapidly growing tumors.

Abstract 576: Immunohistochemistry and radioimaging with hJAA-11 antibody to the Thomsen-Friedenreich antigen: Potential theranostic application for breast cancer

Abstract Theranostics through the utilization of immunohistochemistry followed by radioimaging to determine if metastatic foci will react with a therapeutic antibody will allow for the selection of the patient population that will most benefit from this immunotherapy. The Thomsen-Friedenreich antigen (TF-Ag) has been shown to be involved in ∼90% of carcinomas, specifically breast carcinomas, making it a suitable target for radioimaging and therapy. The anti-TF-Ag antibody, JAA-F11, a mouse monoclonal antibody (mAb), has had success in localization, blocking metastasis, and inhibiting cell proliferation, and binds to ∼80% of breast cancer cell lines, without preference to receptor status. This is significant since the triple negative breast cancer (ER-/PR-/HER2-) has no current targeted treatment. Studies are extended to human breast cancer tissue microarray immunohistochemical (IHC) analysis, which was scored blindly by a pathologist on a semi- quantitative scale. JAA-F11 stained approximately 76% of all breast cancer specimens, which included cases of mucinous, medullary, invasive ductal and neuroendocrine carcinomas. The staining observed was irrespective of receptor status, whereas in normal breast tissue, staining was either absent or very weak/weak. In addition, all available matched lymph node metastasis stained, with greater intensity observed in 39% of cases. JAA-F11 IHC studies performed on human normal organ tissue arrays, showed staining that for the most part was not significantly different from staining obtained with isotype control antibody, or was observed in areas that would not be therapeutically accessible. Furthermore, in an additional IHC study, preliminary results suggest that JAA-F11 significantly stained other carcinomas including those of the colon, bladder, ovary and prostate. In vitro studies show that the humanized JAA-F11 (hJAA-F11) has similar chemical specificity and higher affinity towards the TF-Ag. Imaging studies were performed in a BALB/c mouse breast cancer model with the hJAA-F11 to determine biological reactivity and to predict the feasibility of theranostic imaging prior to therapy. After ∼10 days of tumor growth, mice pretreated with cold iodine water and rabbit IgG to inhibit binding to Fc receptors, were given tail vein injections of hJAA-F11 conjugated with [124]-I. Clear tumor images were obtained up to 144 hours post injection. Biodistribution analysis has provided further results indicating increased%ID/g (7.0±3.9%) in tumor tissue as compared to healthy tissues (brain%ID/g to be 0.21±.09, stomach 0.80±0.19%, and bone 0.90±2.4%). Results support that the hJAA-F11 antibody can be used in a multi-step theranostic approach, with analysis of tumor binding in IHC, followed by imaging to determine in vivo tumor targeting prior to direct immunotherapy or antibody drug conjugate therapy with hJAA-F11. Citation Format: Loukia Karacosta, Holly Johnson, Julia Abdullah, Taylor Chrisikos, Rachel Ludwig, Bradley Turner, Swetha Tati, Diala Ghazal, Munnawar Sajjad, Stephen Koury, Susan Morey, Julie Adams, Kate Rittenhouse-Olson. Immunohistochemistry and radioimaging with hJAA-11 antibody to the Thomsen-Friedenreich antigen: Potential theranostic application for breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 576.

The anti-tumor effect of the quinoline-3-carboxamide tasquinimod: blockade of recruitment of CD11b(+) Ly6C(hi) cells to tumor tissue reduces tumor growth.

BackgroundPrevious work has demonstrated immunomodulatory, anti-tumor, anti-metastatic and anti-angiogenic effects of the small molecule quinoline-3-carboxamide tasquinimod in pre-clinical cancer models. To better understand the anti-tumor effects of tasquinimod in transplantable tumor models, we have evaluated the impact of the compound both on recruitment of myeloid cells to tumor tissue and on tumor-induced myeloid cell expansion as these cells are known to promote tumor development.MethodsMice bearing subcutaneous 4 T1 mammary carcinoma tumors were treated with tasquinimod in the drinking water. A BrdU-based flow cytometry assay was utilized to assess the impact of short-term tasquinimod treatment on myeloid cell recruitment to tumors. Additionally, long-term treatment was performed to study the anti-tumor effect of tasquinimod as well as its effects on splenic myeloid cells and their progenitors. Myeloid cell populations were also immune-depleted by in vivo antibody treatment.ResultsShort-term tasquinimod treatment did not influence the proliferation of splenic Ly6Chi and Ly6Ghi cells, but instead reduced the influx of Ly6Chi cells to the tumor. Treatment with tasquinimod for various periods of time after tumor inoculation revealed that the anti-tumor effect of this compound mainly operated during the first few days of tumor growth. Similar to tasquinimod treatment, antibody-mediated depletion of Ly6Chi cells within that same time frame, caused reduced tumor growth, thereby confirming a significant role for these cells in tumor development. Additionally, long-term tasquinimod treatment reduced the splenomegaly and expansion of splenic myeloid cells during a later phase of tumor development. In this phase, tasquinimod normalized the tumor-induced alterations in myeloerythroid progenitor cells in the spleen but had only limited impact on the same populations in the bone marrow.ConclusionsOur results indicate that tasquinimod treatment reduces tumor growth by operating early after tumor inoculation and that this effect is at least partially caused by reduced recruitment of Ly6Chi cells to tumor tissue. Long-term treatment also reduces the number of splenic myeloid cells and myeloerythroid progenitors, but these effects did not influence established rapidly growing tumors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2481-0) contains supplementary material, which is available to authorized users.

Modulation of Cytokines Production by Indomethacin Acute Dose during the Evolution of Ehrlich Ascites Tumor in Mice.

The aim of the present study was to investigate the influence of a nonselective COX1/COX2 inhibitor (indomethacin) on tumor growth of Ehrlich Ascites Tumor (EAT) in mice, using as parameters the tumor growth and cytokine profile. Mice were inoculated with EAT cells and treated with indomethacin. After 1, 3, 6, 10, and 13 days the animals were evaluated for the secretion of TNFα, IL-1α, IL-2, IL-4, IL-6, IL-10, and IL-13 and PGE2 level in peritoneal cavity. The results have shown that EAT induces PGE2 production and increases tumor cells number from the 10th day. The cytokine profile showed EAT induces production of IL-6 from 10th day and of IL-2 on 13th day; the other studied cytokines were not affected in a significant way. The indomethacin treatment of EAT-bearing mice inhibited the tumor growth and PGE2 synthesis from the 10th day. In addition, the treatment of EAT-bearing mice with indomethacin has stimulated the IL-13 production and has significantly inhibited IL-6 in the 13th day of tumor growth. Taken together, the results have demonstrated that EAT growth is modulated by PGE2 and the inhibition of the tumor growth could be partly related to suppression of IL-6 and induction of IL-13.

The Measure of DAMPs and a role for S100A8 in recruiting suppressor cells in breast cancer lung metastasis

To determine whether there was a relationship between damage associated molecular pattern molecule (DAMP) expression and recruitment of suppressor cells to sites of metastasis we measured relative expression of DAMPs, regulatory T cells (Tregs), and myeloid derived suppressor cells (MDSC) in mice at various stages of breast cancer progression using the 4T1 model. Although S100A8 was expressed at relatively low levels in the tumor cells, expression was 100-fold higher in the lung and liver which are common sites of metastasis for this tumor. Despite the relatively high level of S100A8 expression in the lungs of naïve mice, the level of expression increased further and was significantly elevated after only 7 days of tumor growth. The same pattern was observed for MDSC, and both S100A8 and MDSC expression peaked in the lungs of mice following 21 days of tumor growth. Characterization of MDSC from the lungs revealed expression of RAGE, and the cells were capable of migrating in a dose-dependent manner toward S100A8. In addition, the MDSC expressed low levels of MHC Class I, MHC Class II, CD80, and secreted TGF-β. Taken together, these data suggest that expression of S100A8 in the lungs may facilitate recruitment of MDSC, which may in turn aid in establishing a metastatic niche capable of suppressing a localized immune response.

18F-FDG and 18F-FLT-PET Imaging for Monitoring Everolimus Effect on Tumor-Growth in Neuroendocrine Tumors: Studies in Human Tumor Xenografts in Mice

IntroductionThe mTOR inhibitor everolimus has shown promising results in some but not all neuroendocrine tumors. Therefore, early assessment of treatment response would be beneficial. In this study, we investigated the in vivo and in vitro treatment effect of everolimus in neuroendocrine tumors and evaluated the performance of 18F-FDG and the proliferation tracer 18F-FLT for treatment response assessment by PET imaging.MethodsThe effect of everolimus on the human carcinoid cell line H727 was examined in vitro with the MTT assay and in vivo on H727 xenograft tumors. The mice were scanned at baseline with 18F-FDG or 18F-FLT and then treated with either placebo or everolimus (5 mg/kg daily) for 10 days. PET/CT scans were repeated at day 1,3 and 10.ResultsEverolimus showed significant inhibition of H727 cell proliferation in vitro at concentrations above 1 nM. In vivo tumor volumes measured relative to baseline were significantly lower in the everolimus group compared to the control group at day 3 (126±6% vs. 152±6%; p = 0.016), day 7 (164±7% vs. 226±13%; p<0.001) and at day 10 (194±10% vs. 281±18%; p<0.001). Uptake of 18F-FDG and 18F-FLT showed little differences between control and treatment groups, but individual mean uptake of 18F-FDG at day 3 correlated with tumor growth day 10 (r2 = 0.45; P = 0.034), 18F-FLT mean uptake at day 1 correlated with tumor growth day 7 (r2 = 0.63; P = 0.019) and at day 3 18F-FLT correlated with tumor growth day 7 (r2 = 0.87; P<0.001) and day 10 (r2 = 0.58; P = 0.027).ConclusionEverolimus was effective in vitro and in vivo in human xenografts lung carcinoid NETs and especially early 18F-FLT uptake predicted subsequent tumor growth. We suggest that 18F-FLT PET can be used for tailoring therapy for neuroendocrine tumor patients through early identification of responders and non-responders.