Non-treated Tumors Research Articles

P53-armed oncolytic virotherapy induces abscopal effect in osteosarcoma by promoting immunogenic cell death

Osteosarcoma (OS), the most frequent primary malignant tumor of bone in children and adolescents, is refractory to immune checkpoint inhibitors due to poor antitumor immune response. Chemotherapy and virotherapy induce immunogenic cell death (ICD) and antitumor immune responses, leading to the abscopal effect in untreated tumors. We previously demonstrated the antitumor activity of the telomerase-specific replication-competent oncolytic adenoviruses OBP-301 and p53-armed OBP-702 in human OS cells. Here, we show the therapeutic potential of chemotherapeutic drugs (doxorubicin, cisplatin) and telomerase-specific oncolytic adenoviruses (OBP-301, p53-armed OBP-702) to induce ICD in human OS cells (U2OS, MNNG/HOS, SaOS-2) and murine OS cells (NHOS). OBP-702 induced more profound ICD via secretion of adenosine triphosphate (ATP) and high-mobility group box protein B1 (HMGB1) compared with chemotherapy and OBP-301 in human OS cells. Murine NHOS cells were also more sensitive to OBP-702 than OBP-301. Subcutaneous NHOS tumor models demonstrated that intratumoral injection of OBP-702 significantly increased the tumor infiltration of cytotoxic CD8+ T cells and induced the abscopal effect against non-treated tumors compared with OBP-301. Our results suggest that OBP-702 is a promising antitumor reagent to induce ICD with secretion of ATP and HMGB1 and the abscopal effect against OS.

Comparative study of immune response to local tumor destruction modalities in a murine breast cancer model.

Immunotherapy is revolutionizing the management of multiple cancer types. However, only a subset of patients responds to immunotherapy. One mechanism of resistance is the absence of immune infiltrates within the tumor. In situ vaccine with local means of tumor destruction that can induce immunogenic cell death have been shown to enhance tumor T cell infiltration and increase efficacy of immune checkpoint blockade. Here, we compare three different forms of localize tumor destruction therapies: radiation therapy (RT), vascular targeted photodynamic therapy (VTP) and cryoablation (Cryo), which are known to induce immunogenic cell death, with their ability to induce local and systemic immune responses in a mouse 4T1 breast cancer model. The effects of combining RT, VTP, Cryo with anti-PD1 was also assessed. We observed that RT, VTP and Cryo significantly delayed tumor growth and extended overall survival. In addition, they also induced regression of non-treated distant tumors in a bilateral model suggesting a systemic immune response. Flow cytometry showed that VTP and Cryo are associated with a reduction in CD11b+ myeloid cells (granulocytes, monocytes, and macrophages) in tumor and periphery. An increase in CD8+ T cell infiltration into tumors was observed only in the RT group. VTP and Cryo were associated with an increase in CD4+ and CD8+ cells in the periphery. These data suggest that cell death induced by VTP and Cryo elicit similar immune responses that differ from local RT.

Fibroblast activation protein-targeted near-infrared photoimmunotherapy depletes immunosuppressive cancer-associated fibroblasts and remodels local tumor immunity.

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) play a critical role in tumor immunosuppression. However, targeted depletion of CAFs is difficult due to their diverse cells of origin and the resulting lack of specific surface markers. Near-infrared photoimmunotherapy (NIR-PIT) is a novel cancer treatment that leads to rapid cell membrane damage. In this study, we used anti-mouse fibroblast activation protein (FAP) antibody to target FAP+ CAFs (FAP-targeted NIR-PIT) and investigated whether this therapy could suppress tumor progression and improve tumor immunity. FAP-targeted NIR-PIT induced specific cell death in CAFs without damaging adjacent normal cells. Furthermore, FAP-targeted NIR-PIT treated mice showed significant tumor regression in the CAF-rich tumor model accompanied by an increase in CD8+ tumor infiltrating lymphocytes (TILs). Moreover, treated tumors showed increased levels of IFN-γ, TNF-α, and IL-2 in CD8+ TILs compared with non-treated tumors, suggesting enhanced antitumor immunity. Cancers with FAP-positive CAFs in their TME grow rapidly and FAP-targeted NIR-PIT not only suppresses their growth but improves tumor immunosuppression. Thus, FAP-targeted NIR-PIT is a potential therapeutic strategy for selectively targeting the TME of CAF+ tumors.

Abstract 4156: Monitoring early chemotherapy response in murine pancreatic ductal adenocarcinoma using non-invasive hyperpolarized magnetic resonance spectroscopic imaging with 13C-pyruvate

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a rarely operable cancer with a very poor prognosis. Nearly 100% of affected patients receive one of two alternative first-line chemotherapies during the course of their disease: 1.) Gemcitabine-Abraxane (G/A) or 2.) FOLFIRINOX. Treatment success varies widely between the patients due to individual molecular, structural tumor and metabolic heterogeneity. In this project, we use hyperpolarized 13C-pyruvate magnetic resonance spectroscopic imaging (HP-MRSI) to non-invasively characterize early metabolic changes in murine endogenous PDAC in response to the mentioned chemotherapies. Methods: 18 KPC (Ptf1awt/cre(C)Kraswt/G12D(K)Trp53fl/fl(P)) mice with 74 distinct tumour nodules, visually identified based on T2-weighted anatomical imaging, were used in this study. The conversion of pyruvate to lactate was detected by HP-MRSI, performed on 7 T preclinical system using modified 3D balanced steady-state free precession (bSSFP) sequence on day 0 pre-therapy and on day 3 post-therapy. 100μg/g G (intraperitoneally) and 30μg/g A (intravenously) were applied twice on day 0 post-scan and day 2 (n=6). FOLFIRINOX (60μg/g 5-Fluoruracil, 27μg/g Irinotecan, 12.8μg/g Oxaliplatin, 60μg/g Leucovorin) was injected intravenously once on day 0 post-scan (n=6). Vehicle mice (n=6) were left untreated. Area under the curves (AUC) of lactate to pyruvate time-courses (AUCl/AUCp) were calculated. Primary murine PDAC cell lines were established from biopsies of 58 imaging region, sequenced using 3’RNA-Seq (Poly-ASeq) method and analysed using Dseq2 R package. Results: G/A and FOLFIRINOX-treated tumors exhibited heterogeneous metabolic responses in distinct nodules, while non-treated group tumors remained stable. A significant difference in metabolic changes (p = 0.01, ANOVA) from day 0 to day 3 was observed between FOLFIRINOX, G/A, and non-treated groups. Low glycolytic tumors showed a positive trend, more pronounced with FOLFIRINOX than G/A treatment. Metabolically active nodules with high AUCl/AUCp on day 0 reduced lactate turnover under therapy. These findings suggest that metabolic HP-MRSI can detect treatment-induced changes in tumor composition and subtype-specific epithelial/mesenchymal plasticity, correlating with the metabolic phenotype. Further investigation will include histology, subtype-specific biomarkers, transcriptional signatures, and regional transcriptome analysis to explain molecular changes in metabolism. Conclusions: We have applied a non-invasive approach for detection of metabolic changes under chemotherapy in murine PDAC. These findings will contribute in the long term to the establishment of predictive imaging biomarkers for PDAC. Citation Format: Irina Heid, Anna-Maria Schmidmüller, Simon Baller, Jason G. Skinner, Geoffrey J. Topping, Wolfgang Gottwald, Hussein Trabulssi, Rupert Öllinger, Katja Steiger, Roland Rad, Franz Schilling, Rickmer F. Braren. Monitoring early chemotherapy response in murine pancreatic ductal adenocarcinoma using non-invasive hyperpolarized magnetic resonance spectroscopic imaging with 13C-pyruvate [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 4156.

STING activator 2'3'-cGAMP enhanced HSV-1-based oncolytic viral therapy.

Oncolytic viruses (OVs) can selectively replicate in tumor cells and remodel the microenvironment of immunologically cold tumors, making them a promising strategy to evoke antitumor immunity. Similarly, agonists of the stimulator of interferon genes (STING)-interferon (IFN) pathway, the main cellular antiviral system, provide antitumor benefits by inducing the activation of dendritic cells (DC). Considering how the activation of the STING-IFN pathway could potentially inhibit OV replication, the use of STING agonists alongside OV therapy remains largely unexplored. Here, we explored the antitumor efficacy of combining an HSV-1-based OV, C-REV, with a membrane-impermeable STING agonist, 2'3'-GAMP. Our results demonstrated that tumor cells harbor a largely defective STING-IFN pathway, thereby preventing significant antiviral IFN induction regardless of the permeability of the STING agonist. In vivo, the combination therapy induced more proliferative KLRG1-high PD1-low CD8+ T-cells and activated CD103+ DC in the tumor site and increased tumor-specific CD44+ CD8+ T-cells in the lymph node. Overall, the combination therapy of C-REV with 2'3'-cGAMP elicited antitumor immune memory responses and significantly enhanced systemic antitumor immunity in both treated and non-treated distal tumors.

Loss of the OX40 Target in Lymphoma Patients after Combining an Anti-OX40 Agonist Antibody with in Situ Vaccination

INTRODUCTION:Previous clinical trials have shown that in situ vaccination using the combination of low-dose radiotherapy and intratumoral injection of CpG, a Toll-like receptor 9 (TLR9) agonist, can elicit T cell immune responses in patients with low grade lymphoma and regression at both the treated and non-treated distal tumor sites in patients with low-grade lymphoma (Brody et al., JCO 2010, Frank et al., Cancer Discovery 2018, Hammerich et al., Nat Med 2019). OX40 is a T cell activating co-receptor, belonging to the TNFR superfamily. Ligation of this receptor by an agonist anti-OX40 antibody leads to enhanced T cell activation of effector CD4 T cells while inhibiting Treg function. In preclinical models, addition of an anti-OX40 antibody to intratumoral CpG cured established tumors (Sagiv-Barfi I, et al. Sci Transl Med. 2018). Intratumoral injection of CpG up-regulated OX40 on CD4 T cells in the microenvironment enhancing the binding of agonistic anti-OX40 antibody that triggered a robust immune response. These results provided the rationale for a clinical trial of in situ vaccination (NCT03410901) using a human IgG1 agonistic anti-OX40 antibody (BMS-986178). STUDY:Fourteen patients with low-grade lymphoma received low-dose (2Gy x 2) radiotherapy to a single tumor site followed by 5 weekly intratumoral injections of 2 mg CpG-ODN (SD-101, TriSalus Life Sciences) and 3.75 mg anti-OX40 antibody into the same site. 40mg of anti-OX40 was administered intravenously starting on day 2 and continuing every 4 weeks for a total of 6 infusions. Therapy was well tolerated with no dose-limiting toxicities. Response to therapy was assessed using the revised Lugano criteria (Cheson et al., JCO 2014) based on CT scans at 12, 24, 48, 72, and 96 weeks. Fine needle aspirates (FNA) were performed at both the injected and a non-injected site outside the radiation field prior to, and at 1 and 6 weeks after initiation of treatment. Cells were analyzed by flow cytometry and droplet-based single-cell RNA sequencing to determine population dynamics, response to therapy as well as antibody binding and target occupancy by the anti-OX40 antibody. RESULTS:Of the 14 patients enrolled, 1 patient attained a partial response (PR), while 9 had stable disease (SD) and 4 had disease progression (PD) as their best overall response. Investigation into the binding of the treatment antibody and target occupancy by flow cytometry showed saturation of all the OX40 sites on CD4 T cells by week 6 at both the injected and non-injected tumors. However, further investigation also showed there was significantly less OX40 on the cell surface after treatment, suggesting a loss of the antibody target (Figure 1). This loss was reproduced by in vitro assays where activated blood CD4 T cells exhibited a loss of cell surface OX40 when cultured with BMS-986178. Additionally, Quigley et al ( Clin Cancer Res 2019) previously showed that an OX40 receptor occupancy of greater than 40% led to a loss of target in mice and in blood T cells of solid tumor patients. We further observed that the loss of cell surface OX40 occurred in all CD4 T subsets, including effector CD4 T cells and T Regulatory cells (Tregs). This result contrasted with our in vivo and in vitro findings in successful therapeutic mouse models where we did not observe this loss of cell surface OX40 on effector CD4 T cells after treatment with a mouse-specific anti-OX40 agonist antibody. C ONCLUSION: Our observations suggest that treatment with an agonistic anti-OX40 antibody induced a loss of OX40 on the cell surface of effector CD4 T cells in the tumor microenvironment. Fewer molecules of OX40 receptor may have constrained the efficacy of subsequent anti-OX40 infusions and may explain why the clinical responses observed in this study were lower than observed in our past clinical trials of in situ vaccination.

Anticancer activity of Bacopa monnieri through apoptosis induction and mitophagy-dependent NLRP3 inflammasome inhibition in oral squamous cell carcinoma

BackgroundBacopa monnieri (BM) is traditionally used in human diseases for its antioxidant, anti-inflammatory and neuroprotective effects. However, its anticancer potential has been poorly understood. AimThe aim of this study was to explore the detailed anticancer mechanism of BM against oral cancer and to identify the bioactive BM fraction for possible cancer therapeutics. ResultsWe performed bioactivity-guided fractionation and identified that the aqueous fraction of the ethanolic extract of BM (BM-AF) had a potent anticancer potential in both in vitro and in vivo oral cancer models. BM-AF inhibited cell viability, colony formation, cell migration and induced apoptotic cell death in Cal33 and FaDu cells. BM-AF at low doses promoted mitophagy and BM-AF mediated mitophagy was PARKIN dependent. In addition, BM-AF inhibited arecoline induced reactive oxygen species production in Cal33 cells. Moreover, BM-AF supressed arecoline-induced NLR family pyrin domain containing 3 (NLRP3) inflammasome activation through mitophagy in Cal33 cells. The in vivo antitumor effect of BM-AF was further validated in C57BL/6J mice through a 4-nitroquinolin-1-oxide and arecoline-induced oral cancer model. The tumor incidence was significantly reduced in the BM-AF treated group. Further, data obtained from western blot and immunohistochemistry analysis showed increased expression of apoptotic markers and decreased expression of inflammasome markers in the tongue tissue obtained from BM-AF treated mice in comparison with the non-treated tumor bearing mice. ConclusionIn conclusion, BM-AF exhibited potent anticancer activity through apoptosis induction and mitophagy-dependent inhibition of NLRP3 inflammasome activation in both in vitro and in vivo oral cancer models. Moreover, we have investigated apoptosis and mitophagy-inducing compounds from this plant extract having anticancer activity against oral cancer cells.

Super2-IL-33 armored CAR T cells reshape the tumor microenvironment to universally suppress solid tumor growth

Abstract Tumor associated immunosuppressive cells limit the efficacy of CAR T cells against solid tumors. To alter immunosuppression in the tumor microenvironment, CAR T cells were armored with an IL-2 superkine (Super2) and alarmin IL-33 and adoptively transferred into immunocompetent mice with established primary or metastatic B16F10 melanoma or intradermal MC38 colon carcinoma. We show that Super2-IL-33 CAR T cells universally promoted the control of solid tumors in mice. Analysis of tumor infiltrating lymphocytes (TILs) revealed that Super2 and IL-33 expanded CAR T cells and endogenous lymphocytes cells, resulting in an increased ratio of CD8 effector T cells to regulatory T cells. The expression of the CAR or effector molecules was dispensable for CAR T cell-induced tumor control, underscoring the contribution of endogenous immune cells in mediating therapeutic efficacy. We next evaluated the effects of Super2-IL-33 CAR T cells in bone, liver, and thymus (BLT) humanized mice to assess efficacy against human tumors in an immunocompetent model. BLT mice were intradermally inoculated with Caki1, a human renal cell carcinoma in which non-treated tumors are highly enriched for regulatory T cells. To determine the impact of Super2-IL-33 armored CAR T cells on human tumors and infiltrating immune cells, tumor growth and TILs were analyzed. Super2-IL-33 armored human CAR T cells recruit endogenous immune cells to the tumor that facilitate the suppression of tumor growth. Thus, the Super2-IL-33 cytokine combination may serve as a universal platform capable of overcoming immunosuppression to promote broad anti-tumor immunity. Supported by grants from NIH (R43-CA271880 and R01-CA254042)

Chronic effects of pulsed high intensity focused ultrasound aided delivery of gemcitabine in a mouse model of pancreatic cancer

Pulsed high intensity focused ultrasound (pHIFU) is a non-invasive method that allows to permeabilize pancreatic tumors through inertial cavitation and thereby increase the concentration of systemically administered drug. In this study the tolerability of weekly pHIFU-aided administrations of gemcitabine (gem) and their influence on tumor progression and immune microenvironment were investigated in genetically engineered KrasLSL.G12D/þ; p53R172H/þ; PdxCretg/þ (KPC) mouse model of spontaneously occurring pancreatic tumors. KPC mice were enrolled in the study when the tumor size reached 4–6 mm and treated once a week with either ultrasound-guided pHIFU (1.5 MHz transducer, 1 ms pulses, 1% duty cycle, peak negative pressure 16.5 MPa) followed by administration of gem (n = 9), gem only (n = 5) or no treatment (n = 8). Tumor progression was followed by ultrasound imaging until the study endpoint (tumor size reaching 1 cm), whereupon the excised tumors were analyzed by histology, immunohistochemistry (IHC) and gene expression profiling (Nanostring PanCancer Immune Profiling panel). The pHIFU + gem treatments were well tolerated; the pHIFU-treated region of the tumor turned hypoechoic immediately following treatment in all mice, and this effect persisted throughout the observation period (2–5 weeks) and corresponded to areas of cell death, according to histology and IHC. Enhanced labeling by Granzyme-B was observed within and adjacent to the pHIFU treated area, but not in the non-treated tumor tissue; no difference in CD8 + staining was observed between the treatment groups. Gene expression analysis showed that the pHIFU + gem combination treatment lead to significant downregulation of 162 genes related to immunosuppression, tumorigenesis, and chemoresistance vs gem only treatment.

Abstract No. 158 Characterizing the Anti-Tumor Immune Response to IRE vs Thermal Ablation Therapy in an Immunocompetent Oncopig Model of Hepatocellular Carcinoma

Immunotherapies for cancer treatment are designed to mobilize a patient’s own immune system against their malignancy locally and systemically. Percutaneous ablation techniques have the potential to work in conjunction with modern immunotherapies by priming the patient’s own lymphocytes to attack previously untreated cancer cells. This phenomenon, known as the abscopal effect, has garnered interest because of the potential for locoregional therapies to induce local and systemic anti-tumor effects and shrinkage of distant tumors (Fionda, 2020). The goal of this study is to evaluate the anti-tumor immune response to irreversible electroporation (IRE) compared with microwave (MWA) and cryoablation in an immunocompetent Oncopig liver tumor model of hepatocellular carcinoma. In-situ liver tumors were induced in a transgenic immunocompetent porcine liver model (2-4 tumors in each liver) with Cre-recombinase induced TP53 and KRAS mutations (Oncopig). Oncopigs were separated into three treatment groups (n = 2), each undergoing either MWA, cryoablation or IRE treatment of a single liver tumor under US or CT guidance. Pre-, immediate post- and 1-week post ablation imaging was performed to evaluate treated and non-treated tumor growth. At each time point, venous blood was also drawn and assayed for critical cytokines related to anti- or pro-tumor immune response. Systemic cytokine levels were correlated to targeted and off-targeted tumor volumes after each time point. Liver tumors were induced at an 83.3% (20/24) success rate. Treated tumors decreased by an average 7% in volume across all treatment modalities on 1-week follow-up imaging. Off-target tumors increased by volume by 71.7% with IRE, 13.6% with MWA and 1.4% with cryoablation. On 1-week blood samples, IRE treated pigs demonstrated lower circulating concentrations of pro-inflammatory IL-2 cytokines when compared with cryoablation (10.9 vs 40.7 pg/mL; P = 0.051). There was no difference in IL-2 levels between MWA and cryoablation-treated pigs (40.7 vs 42.4 pg/mL; P = 0.97). The pro- and anti-tumor immune response is dependent on the type of ablation modality. IRE had the lowest levels of inflammatory marker IL-2 and the lowest degree of tumor control in off target tumors at the one-week time point. Modulating inflammatory pathways via these cytokines after IRE may potentially play a role in amplifying the abscopal effect in liver tumors.

Photothermic therapy with cuttlefish ink-based nanoparticles in combination with anti-OX40 mAb achieve remission of triple-negative breast cancer.

Immunostimulatory monoclonal antibodies (IS-mAb) have been proven to enhance the therapeutic effectiveness of various anticancer therapy. In the present investigation, we launched a separate combinational therapy for the treatment of triple-negative breast cancer (TNBC) using cuttlefish ink-based nanoparticles (CINPs) for photothermal therapy (PTT) and anti-OX40 antibody. Our goal was to increase the therapeutic response to the disease. CINPs were characterized by their physicochemical properties, which revealed that they had a hydrodynamic diameter ranging from 128 to 148nm, a negative surface charge, and a high photothermal conversion efficiency under both in vitro and in vivo settings. In TNBC model, we evaluated the therapeutic effectiveness of the following groups: CINP-PTT+anti-OX40 Ab (G1), CINPs-PTT (G2), CINPs+anti-OX40 Ab (G3), anti-OX40 (G4) or PBS (G5). In each case, we assessed the efficacy of these groups against one another. The intratumor administration of all of the substances and therapies was performed. CINP-PTT+anti-OX40 Ab and CINP+anti-OX40 Ab (particularly CINP-PTT+anti-OX40 Ab) induced significant tumor regression in treated (breast) and non-treated (flank) tumor, and completely inhibited lung metastasis, thereby inducing a higher survival rate in mice in comparison to CINP-PTT, anti-OX40 Ab, or PBS. This was the case because in CINPs-treated tumors, particularly those treated with CINPs-PTT, intratumoral injection of CINPs increased the frequency of OX40, CD8 double-positive T cells. CINPs improved the conversion of the macrophage phenotype from M2 to M1 in vitro, which is significant from an immunological point of view. In addition, anti-OX40 Ab combined with CINPs or, more specifically, CINPs-PPT produced a larger frequency of preexisting and newly formed tumor-specific CD8 T cells, as well as an enhanced frequency of CD8 T cells infiltrating non-treated tumors, in comparison to respective monotherapies. When the data were taken into consideration as a whole, it seemed that CINPs-based PTT may effectively enhance the antitumor response effectiveness of anti-OX40 Ab.

Attenuation of Tumor Burden in Response to Rucaparib in Lung Adenocarcinoma: The Contribution of Oxidative Stress, Apoptosis, and DNA Damage.

In cancer, overactivation of poly (ADPribose) polymerases (PARP) plays a relevant role in DNA repair. We hypothesized that treatment with the PARP inhibitor rucaparib may reduce tumor burden via several biological mechanisms (apoptosis and oxidative stress) in mice. In lung tumors (LP07 lung adenocarcinoma) of mice treated/non-treated (control animals) with PARP inhibitor (rucaparib,150 mg/kg body weight/24 h for 20 day), PARP activity and expression, DNA damage, apoptotic nuclei, cell proliferation, and redox balance were measured using immunoblotting and immunohistochemistry. In lung tumors of rucaparib-treated mice compared to non-treated animals, tumor burden, PARP activity, and cell proliferation decreased, while DNA damage, TUNEL-positive nuclei, protein oxidation, and superoxide dismutase content (SOD)2 increased. In this experiment on lung adenocarcinoma, the pharmacological PARP inhibitor rucaparib elicited a significant improvement in tumor size, probably through a reduction in cell proliferation as a result of a rise in DNA damage and apoptosis. Oxidative stress and SOD2 also increased in response to treatment with rucaparib within the tumor cells of the treated mice. These results put the line forward to the contribution of PARP inhibitors to reduced tumor burden in lung adenocarcinoma. The potential implications of these findings should be tested in clinical settings of patients with lung tumors.

Advantage of extracellular vesicles in hindering the CD47 signal for cancer immunotherapy.

The cluster of differentiation 47 (CD47) protein is abundantly expressed on various malignant cells and suppresses the phagocytic function of macrophages and dendritic cells. High CD47 expression levels are correlated with poor cancer survival. Antagonizing CD47 antibodies with potent antitumor effects have been developed in clinical trials, but have critical side effects, inducing anemia and thrombocytopenia. To develop a safe and potent CD47 blockade, we designed extracellular vesicles (EVs) harboring signal regulatory protein alpha (SIPRα)-EV-SIRPα (EVs that express SIPRα). EV-SIRPα showed minimal toxic effects on hematologic parameters and utilized RBCs as delivery vehicles to tumors rather than inducing anemia. EV-SIRPα inhibited ligation of residual CD47 molecules, which attribute to the EV-endocytosis-mediated CD47 depletion and steric hindrance of EV. In an immunologically cold tumor model, EV-SIRPα induced tumor-specific T-cell-mediated antitumor effects. When directly administered to the accessible lesions, EV-SIRPα monotherapy elicited an abscopal effect in the B16F10 tumor model by increasing immune cell infiltration and CD8⁺-mediated immunity against non-treated tumors. The combinational approach by loading doxorubicin into the EV-SIRPα dramatically reduced the tumor burden and led to 80% complete remission rate. Thus, a potent EV-based CD47 blockade that is hematologically safe, has efficient signaling blocking efficacy, and has systemic antitumor immunity against cancer is recommended.

In high-grade ovarian carcinoma, platinum-sensitive tumor recurrence and acquired-resistance derive from quiescent residual cancer cells that overexpress CRYAB, CEACAM6, and SOX2.

Most high-grade ovarian carcinomas (HGOCs) are sensitive to carboplatin (CBP)-based chemotherapy but frequently recur within 24 months. Recurrent tumors remain CBP-sensitive and acquire resistance only after several treatment rounds. Recurrences arise from a small number of residual tumor cells not amenable to investigation in patients. We developed patient-derived xenografts (PDXs) that allow the study of these different stages of CBP-sensitive recurrence and acquisition of resistance. We generated PDX models from CBP-sensitive and intrinsically resistant HGOC. PDXs were CBP- or mock-treated and tumors were sampled, after treatment and at recurrence. We also isolated models with acquired-resistance from CBP-sensitive PDXs. Tumors were characterized at the histological and transcriptome levels. PDX models reproduced treatment response seen in the patients. CBP-sensitive residual tumors contained nonproliferating tumor cell clusters embedded in a fibrotic mesh. In nontreated PDX tumors and treated CBP-resistant tumors, fibrotic tissue was not prevalent. Residual tumors had marked differences in gene expression when compared to naïve and recurrent tumors, indicating downregulation of the cell cycle and proliferation and upregulation of interferon response and the epithelial-mesenchymal transition. This gene expression pattern resembled that described in embryonal diapause and 'drug-tolerant persister' states. Residual and acquired-resistance tumors share the overexpression of three genes: CEACAM6, CRYAB, and SOX2. Immunostaining analysis showed strong CEACAM6, CRYAB, and SOX2 protein expression in CBP-sensitive residual and acquired-resistance PDX, thus confirming the RNA profiling results. In HGOC PDX, CBP-sensitive recurrences arise from a small population of quiescent, drug-tolerant, residual cells embedded in a fibrotic mesh. These cells overexpress CEACAM6, CRYAB, and SOX2, whose overexpression is also associated with acquired resistance and poor patient prognosis. CEACAM6, CRYAB, and SOX2 may thus serve as a biomarker to predict recurrence and emergence of resistant disease in CBP-treated HGOC patients. © 2022 The Pathological Society of Great Britain and Ireland.

Intratumoral Electroporation of Plasmid Encoded IL12 and Membrane-Anchored Anti-CD3 Increases Systemic Tumor Immunity.

This DNA-encodable polyclonal T-cell stimulator (membrane-anchored anti-CD3 plasmid) may represent a key addition to intratumoral IL12 therapies in the clinic.

Tissue Distribution, Histopathological and Genotoxic Effects of Magnetite Nanoparticles on Ehrlich Solid Carcinoma

Nanoparticles can potentially cause adverse effects on cellular and molecular level. The present study aimed to investigate the histopathological changes and DNA damage effects of magnetite nanoparticles (MNPs) on female albino mice model with Ehrlich solid carcinoma (ESC). Magnetite nanoparticles coated with L-ascorbic acid (size ~ 25.0 nm) were synthesized and characterized. Mice were treated with MNPs day by day, intraperitoneally (IP), intramuscularly (IM), or intratumorally (IT). Autopsy samples were taken from the solid tumor, thigh muscle, liver, kidney, lung, spleen, and brain for assessment of iron content, histopathological examination, and genotoxicity using comet assay. The liver, spleen, lung, and heart had significantly higher iron content in groups treated IP. On the other hand, tumor, muscles, and the liver had significantly higher iron content in groups treated IT. MNPs induced a significant DNA damage in IT treated ESC. While a significant DNA damage was detected in the liver of the IP treated group, but no significant DNA damage could be detected in the brain. Histopathological findings in ESC revealed a marked tumor necrosis, 50% in group injected IT but 40% in group injected IP and 20% only in untreated tumors. Other findings include inflammatory cell infiltration, dilatation, and congestion of blood vessels of different organs of treated groups in addition to appearance of metastatic cancer cells in the liver of non-treated tumor group. MNPs could have an antitumor effect but it is recommended to be injected intratumorally to be directed to the tumor tissues and reduce its adverse effects on healthy tissues.

Cyclin-dependent kinase 4/6 inhibitors suppress tumor growth in extramammary Paget's disease.

Extramammary Paget’s disease (EMPD) is a rare adnexal neoplasm commonly seen in the genital areas among the senior population. The prognosis of advanced EMPD is not favorable; thus, the development of potential treatments has long been sought. Cyclin‐dependent kinase (CDK) 4/6 inhibitors such as abemaciclib and palbociclib have been proven effective against metastatic breast cancer; however, no studies have addressed CDK4/6 inhibitors as an EMPD treatment. We herein examine the efficacy of CDK4/6 inhibitors against an EMPD patient‐derived xenograft (PDX) model. Abemaciclib (50 mg/kg/day) or palbociclib (120 mg/kg/day) was given orally to tumor‐bearing NOD/Scid mice over a 3‐week period. We also investigated the protein expression levels of CDK4/6 and cyclin D1 through immunohistochemical staining using EMPD clinical samples. Treatment with abemaciclib or palbociclib as a single agent was found to significantly suppress tumor growth in EMPD‐PDX. The Ki‐67‐positive ratio of the treated EMPD‐PDX tumors was significantly lower than that of the nontreated tumors. Clinically, the expression levels of CDK4 and cyclin D1 were significantly higher in the EMPD tumor cells than in the normal epidermis. Our results suggest that CDK4/6 inhibitors could be novel and potent therapeutics for the treatment of EMPD.

Serial FNA allows direct sampling of malignant and infiltrating immune cells in patients with B-cell lymphoma receiving immunotherapy.

Fine-needle aspiration (FNA) is used to diagnose malignancies, recurrences, and metastases. The procedure is quick and well tolerated and can be facilitated by ultrasound guidance. This article describes the authors' experience in using serial FNA to harvest cellular material during 4 clinical trials of immunotherapy by in situ vaccination in patients with low-grade lymphoma. Two hundred ninety-six FNA samples were collected from 44 patients over a span of approximately 6 weeks for each patient. Samples were sufficient in quantity and quality to be analyzed by flow cytometry and/or single-cell messenger RNA sequencing. FNA samples yielded an average of 12 × 106 cells with a mean cellular viability of 86%. Material collected from the tumor lymph nodes differed significantly in the proportions and phenotypes of cellular populations in comparison with matched peripheral blood samples. A comparison of flow cytometry results obtained by FNA directly from the patient and by FNA performed ex vivo and a dissociation of the same lymph node after surgical excision confirmed that FNA sampling of the patient accurately represented the tumor and the microenvironment. An analysis of the FNA samples from immunotherapy-treated target lymph nodes versus nodes from nontreated tumor sites provided insight into the impact of specific immunotherapy regimens. This is the largest study describing the use of serial FNA sampling to harvest cellular material during immunotherapy clinical trials. The success of this technique opens the door for FNA sampling to expand significantly future investigations of the dynamic effects of investigational agents, be they immunotherapies or targeted therapies.

Value of 18F-FDG PET/MR Imaging in the Early Evaluation of Treatment Response following Radiofrequency Ablation of Liver Cancer in a Rabbit Model

PurposeTo test the hypothesis that 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) and magnetic resonance (MR) imaging can detect early residual tumor following radiofrequency (RF) ablation of liver cancer using a VX2 tumor model. Materials and MethodsTwenty-four rabbits with VX2 liver tumors were randomly divided into 3 groups (n = 8/group): Group 1 without RF ablation treatment, Group 2 with complete ablation, and Group 3 with partial ablation. An 18F-FDG PET/MR imaging scan was obtained within 2 hours after RF ablation. The maximum standardized uptake values (SUV) of the nontreated liver tumor, benign periablational enhancement (BPE), residual tumor, ablated tumor, and adjacent liver parenchyma and mean SUV of the normal liver were measured. The ratios of maximum SUV for these targets to the mean SUV of the normal liver (TNR) were calculated and compared. ResultsThe mean TNR of the nontreated liver tumors in Group 1 was significantly greater than that of the adjacent liver parenchyma (8.68 ± 0.71 vs 1.89 ± 0.26, P < .001). In Group 2, the mean TNR of BPE was significantly greater than that of the adjacent liver parenchyma (2.85 ± 0.20 vs 1.86 ± 0.25, P < .001). In Group 3, the mean TNR of the residual tumor was significantly greater than that of BPE (8.64 ± 0.59 vs 2.78 ± 0.23, P < .001), which was significantly greater than that of completely ablated tumor (2.78 ± 0.23 vs 0.50 ± 0.06, P < .001). Conclusions18F-FDG PET/MR imaging may serve as a promising imaging tool for the early detection of viable residual tumors due to incomplete tumor ablation.

MTL-CEBPA Combined with Immunotherapy or RFA Enhances Immunological Anti-Tumor Response in Preclinical Models.

The transcription factor CEBPA is a master regulator of liver homeostasis, myeloid cell differentiation and is downregulated in several oncogenic diseases. MTL-CEBPA is a small activating RNA drug which upregulates gene expression of CEBPA for treatment of hepatocellular carcinoma (HCC). We investigate whether MTL-CEBPA has immune modulatory effects by combining MTL-CEBPA with an anti-PD-1 checkpoint inhibitor (CPI) and/or radiofrequency ablation (RFA) in two preclinical models. First, mice with two flanks of HCC tumors (BNL) were treated with combinations of RFA (right flank), anti-PD-1 or MTL-CEBPA. The reduction of the left flank tumors was most pronounced in the group treated with RFA+anti-PD1+MTL-CEBPA and 7/8 animals responded. This was the only group with a significant increase in CD8+ and CD49b+/CD45+ tumor infiltrating lymphocytes (TIL). Second, a combination of anti-PD-1+MTL-CEBPA was tested in a CT26 colon cancer model and this treatment significantly reduced tumor size, modulated the tumor immune microenvironment and increased TILs. These data suggest a clinical role for combination treatment with CPIs, RFA and MTL-CEBPA through synergistic priming of the immune tumor response, enabling RFA and CPIs to have a pronounced anti-tumor effect including activity in non-treated tumors in the case of RFA.