Metastatic disease is the major cause of cancer death, and the lung is one of the most common sites of cancer metastases. To investigate systemic antitumor effects or protective potential of local ...therapies, mouse models with induced metastases are indispensable in preclinical cancer research. Here, we describe the protocol for the metastatic mouse model established through induced 4T1 mammary carcinoma metastases. With minor prior optimization, it can be applied to other tumor cell lines of interest.
In calcium electroporation (CaEP), electroporation enables the cellular uptake of supraphysiological concentrations of Ca
, causing the induction of cell death. The effectiveness of CaEP has already ...been evaluated in clinical trials; however, confirmatory preclinical studies are still needed to further elucidate its effectiveness and underlying mechanisms. Here, we tested and compared its efficiency on two different tumor models to electrochemotherapy (ECT) and in combination with gene electrotransfer (GET) of a plasmid encoding interleukin-12 (IL-12). We hypothesized that IL-12 potentiates the antitumor effect of local ablative therapies as CaEP and ECT.
The effect of CaEP was tested
as well as
in murine melanoma B16-F10 and murine mammary carcinoma 4T1 in comparison to ECT with bleomycin. Specifically, the treatment efficacy of CaEP with increasing calcium concentrations alone or in combination with IL-12 GET in different treatment protocols was investigated. We closely examined the tumor microenvironment by immunofluorescence staining of immune cells, as well as blood vessels and proliferating cells.
, CaEP and ECT with bleomycin reduced cell viability in a dose-dependent manner. We observed no differences in sensitivity between the two cell lines. A dose-dependent response was also observed
; however, the efficacy was better in 4T1 tumors than in B16-F10 tumors. In 4T1 tumors, CaEP with 250 mM Ca resulted in more than 30 days of growth delay, which was comparable to ECT with bleomycin. In contrast, adjuvant peritumoral application of IL-12 GET after CaEP prolonged the survival of B16-F10, but not 4T1-bearing mice. Moreover, CaEP with peritumoral IL-12 GET modified tumor immune cell populations and tumor vasculature.
Mice bearing 4T1 tumors responded better to CaEP
than mice bearing B16-F10 tumors, even though a similar response was observed
. Namely, one of the most important factors might be involvement of the immune system. This was confirmed by the combination of CaEP or ECT with IL-12 GET, which further enhanced antitumor effectiveness. However, the potentiation of CaEP effectiveness was also highly dependent on tumor type; it was more pronounced in poorly immunogenic B16-F10 tumors compared to moderately immunogenic 4T1 tumors.
Multimodal treatment approaches, such as radio-immunotherapy, necessitate regimen optimization and the investigation of the interactions of different modalities. The aim of this study was two-fold. ...Firstly, to select the most effective combination of irradiation and the previously developed tumor cell-based vaccine and then to provide insight into the immune response to the selected combinatorial treatment. The study was performed in immunologically different murine tumor models: B16F10 melanoma and CT26 colorectal carcinoma. The most effective combinatorial treatment was selected by comparing three different IR regimens and three different vaccination regimens. We determined the local immune response by investigating immune cell infiltration at the vaccination site and in tumors. Lastly, we determined the systemic immune response by investigating the amount of tumor-specific effector lymphocytes in draining lymph nodes. The selected most effective combinatorial treatment was 5× 5 Gy in combination with concomitant single-dose vaccination (B16F10) or with concomitant multi-dose vaccination (CT26). The combinatorial treatment successfully elicited a local immune response at the vaccination site and in tumors in both tumor models. It also resulted in the highest amount of tumor-specific effector lymphocytes in draining lymph nodes in the B16F10, but not in the CT26 tumor-bearing mice. However, the amount of tumor-specific effector lymphocytes was intrinsically higher in the CT26 than in the B16F10 tumor model. Upon the selection of the most effective combinatorial treatment, we demonstrated that the vaccine elicits an immune response and contributes to the antitumor efficacy of tumor irradiation. However, this interaction is multi-faceted and appears to be dependent on the tumor immunogenicity.
Biliary tract cancers (BTCs) are usually diagnosed at an advanced stage, when the disease is incurable. Currently used tumor biomarkers have limited diagnostic value for BTCs, so there is an urgent ...need for sensitive and specific biomarkers for their earlier diagnosis. Deregulation of the homeostasis of trace elements is involved in the carcinogenesis of different cancers, including BTCs. The objective of the study is to determine/compare the total concentrations of copper (Cu), zinc (Zn) and iron (Fe) and the proportions of free Cu and Cu bound to ceruloplasmin (Cp) and the isotopic ratio of
Cu/
Cu in serum samples from healthy volunteers and cancer patients using inductively coupled plasma-mass spectrometry-based methods (ICP-MS).
In this prospective, noninterventional, nonrandomized study 20 patients and 20 healthy volunteers will be enrolled to identify serum Cu, Zn and Fe levels, Cu isotopic fractionation as a predictive biomarker of response to systemic therapy of BTCs, which will be evaluated by computed tomography. Newly developed analytical methods based on ICP-MS will be applied to metal-based biomarker research in oncology.
In the study the comparison of the total concentration of selected trace elements, the proportion of free Cu and Cu bound to Cp and the isotopic ratio of
Cu/
Cu in serum samples from healthy volunteers and cancer patients will be conducted to provide the foundation for the development of a BTC cancer screening methodology and the data on their usability as a potential predictive biomarker for BTCs of response to systemic therapy.
Electrochemotherapy (ECT) is a clinically acknowledged method that combines the use of anticancer drugs and electrical pulses. Electrochemotherapy with bleomycin (BLM) can induce immunogenic cell ...death (ICD) in certain settings. However, whether this is ubiquitous over different cancer types and for other clinically relevant chemotherapeutics used with electrochemotherapy is unknown. Here, we evaluated in vitro in the B16-F10, 4T1 and CT26 murine tumor cell lines, the electrochemotherapy triggered changes in the ICD-associated damage-associated molecular patterns (DAMPs): Calreticulin (CRT), ATP, High Mobility Group Box 1 (HMGB1), and four immunologically important cellular markers: MHCI, MHC II, PD-L1 and CD40. The changes in these markers were investigated in time up to 48 h after ECT. We showed that electrochemotherapy with all three tested chemotherapeutics induced ICD-associated DAMPs, but the induced DAMP signature was cell line and chemotherapeutic concentration specific. Similarly, electrochemotherapy with CDDP, OXA or BLM modified the expression of MHC I, MHC II, PD-L1 and CD40. The potential of electrochemotherapy to change their expression was also cell line and chemotherapeutic concentration specific. Our results thus put the electrochemotherapy with clinically relevant chemotherapeutics CDDP, OXA and BLM on the map of ICD inducing therapies.
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•IL-12 GET locally potentiates the antitumor response of ECT.•Treatment of tumors with ECT + IL-12 GET does not cause unwanted severe side effects.•ECT + GET i.t. therapy induces an ...antitumor immune response at the systemic level.•Higher expression of PDL-1 is associated with poorer response after ECT + IL-12 GET.•Treatment response should be assessed at least 2 months after treatment.
The combined treatment of electrochemotherapy (ECT) and interleukin-12 (IL-12) gene electrotransfer (GET) has already been used in clinical studies in dogs to treat various histological types of spontaneous tumors. The results of these studies show that the treatment is safe and effective. However, in these clinical studies, the routes of administration of IL-12 GET were either intratumoral (i.t.) or peritumoral (peri.t.). Therefore, the objective of this clinical trial was to compare the two IL-12 GET routes of administration in combination with ECT and their contribution to the enhanced ECT response. Seventy-seven dogs with spontaneous mast cell tumors (MCTs) were divided into three groups: one treated with a combination of ECT + GET peri. t. (29 dogs), the second with the combination of ECT + GET i.t. (30 dogs), and the third with ECT alone (18 dogs). In addition, immunohistochemical studies of tumor samples before treatment and flow cytometry of peripheral blood mononuclear cells (PBMCs) before and after treatment were performed to determine any immunological aspects of the treatment. The results showed that local tumor control was significantly better in the ECT + GET i.t. group (p < 0.050) than in the ECT + GET peri.t. or ECT groups. In addition, disease-free interval (DFI) and progression-free survival (PFS) were significantly longer in the ECT + GET i.t. group than in the other two groups (p < 0.050). The data on local tumor response, DFI, and PFS were consistent with immunological tests, as we detected an increased percentage of antitumor immune cells in the blood after treatment in the ECT + GET i.t. group, which also indicated the induction of a systemic immune response. In addition, we did not observe any unwanted severe or long-lasting side effects. Finally, due to the more pronounced local response after ECT + GET i.t., we suggest that treatment response assessment should be performed at least two months after treatment, which meets the iRECIST criteria.
Genska terapija postaja čedalje bolj zanimiva tudi v onkologiji. Med aplikacijami je morda najzanimivejša imunostimulacija. Pripravimo lahko plazmidno DNA, ki nosi zapis za različne imunostimulatorne ...molekule, ki jih vnesemo v celice tumorjev ali normalnih tkiv. Ta tkiva postanejo proizvajalci teh molekul, ki lahko delujejo lokalno ali pa se izločajo tudi sistemsko v krvni obtok. Ker plazmidna DNA ne prehaja celične membrane, so potrebni dostavni sistemi, virusni ali nevirusni. V naših študijah uporabljamo predvsem nevirusni dostavni sistem – elektroporacijo. Interlevkin 12 (IL-12) je eden od zanimivih citokinov, za katerega je znano protitumorsko delovanje s spodbujanjem imunskega odziva in antiangiogenim delovanjem. Namen projekta SmartGene.si je bil pripraviti plazmid z zapisom za interlevkin 12 (plazmid phIL12) in pripraviti vse potrebno za njegovo klinično testiranje za zdravljenje kožnih tumorjev. V konzorciju smo združili moči s partnerji z akademskega in industrijskega področja. Treba je bilo pripraviti plazmid za uporabo v humani onkologiji po zahtevah Evropske agencije za zdravila (EMA). Za prijavo klinične študije na Javno agencijo za zdravila in medicinske pripomočke (JAZMP) smo morali izvesti tudi vse neklinične raziskave o varnosti in učinkovitosti zdravila. Nato je bilo treba razviti postopek priprave zdravila, zagotoviti primerne prostore za pripravo in izvedbo postopka priprave zdravila. V treh letih smo dosegli vse te zastavljene cilje in dobili dovoljenje za izvajanje klinične študije na kožnih tumorjih, ki ga je izdala JAZMP na osnovi pozitivnega mnenja Komisije Republike Slovenije za medicinsko etiko. Zdaj poteka klinična študija faze I preizkušanja plazmida phIL12 na kožnih tumorjih glave in vratu z namenom preveriti varnost in sprejemljivost genskega elektroprenosa plazmida v tumorje. Cilj študije je prav tako določiti primeren odmerek zdravila, ki bi ga v nadaljnji klinični študiji uporabili kot adjuvantno zdravljenje k ablativnim terapijam, kot sta radioterapija ali elektrokemoterapija.
Abstract
When cells or tissues are exposed to an electric field, structural changes can be induced in the cell membrane, allowing molecules to flow into the cells. This condition is useful for ...delivering cytotoxic drugs whose transport into cells is impeded, such as bleomycin and cisplatin. This is referred to as electrochemotherapy (ECT). When DNA or RNA molecules are transported into cells, the approach is called gene electrotransfer (GET). ECT is an effective local treatment for tumors, but it lacks the systemic component of treatment that is essential for fighting metastatic disease. Cancer progression and metastasis are closely related to an inadequate antitumor immune response. Therefore, the antitumor efficacy of interleukin 12 (IL -12), a cytokine that stimulates the innate and adaptive immune response, has been tested in preclinical studies using plasmid DNA encoding IL -12 delivered into the tumor by gene electrotransfer (IL -12 GET). In addition, the combined treatment of ECT and IL -12 GET has already been shown to be effective in the treatment of mouse tumors and has been used in clinical trials in companion dogs to treat various histological types of spontaneous tumors. The results of these studies showed that the treatment is safe and effective. However, in these clinical trials, IL -12 GET was administered by different routes, either intratumorally (i.t.) or peritumorally (peri.t.). Therefore, the aim of this study was to compare the two IL -12 GET routes of administration in combination with ECT and to determine their contribution to the enhanced response to ECT. In the clinical trial, 59 dogs with spontaneous mast cell tumors (MCT) were divided into two groups: one group was treated with the combination of ECT and GET peri.t. (29 dogs) and the other with the combination of ECT + GET i.t. (30 dogs). The efficacy of the treatment was compared with the group of 18 dogs treated with ECT alone. In addition, immunohistochemical studies of tumor samples before treatment and flow cytometric studies of PMBC before and after treatment were performed to determine immunological aspects of the treatment. The results of this study showed that local tumor control was significantly better in the ECT + GET i.t. group (p < 0.05) than in the ECT + GET peri.t. and ECT groups. In addition, the disease-free interval (DFI) and progression-free survival (PFS) were significantly longer in the ECT + GET i.t. group than in the other two groups (p < 0.05). The data on local tumor response, DFI and PFS were consistent with immunological studies, as we detected increased infiltration of antitumor immune cells after treatment in the ECT + GET i.t. group, also suggesting a systemic effect.
Citation Format: Maja Cemazar, Ursa Lampreht Tratar, Nina Milevoj, Katarina Znidar, Katja Ursic Valentinuzzi, Andreja Brozic, Gregor Sersa, Natasa Tozon. Electrochemotherapy and IL-12 gene electrotransfer in treatment of mast cell tumors in companion dogs. abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5122.
Cancer immunotherapy is a rapidly developing field, with numerous drugs and therapy combinations waiting to be tested in pre-clinical and clinical settings. However, the costly and time-consuming ...trial-and-error approach to development of new treatment paradigms creates a research bottleneck, motivating the development of complementary approaches. Computational modelling is a compelling candidate for this task, however, difficulties associated with the validation of such models limit their use in pre-clinical and clinical settings. Here we propose a bottom-up deterministic computational model to simulate tumour response to treatment with anti-programmed-death-1 antibodies (anti-PD-1). The model was built with validation in mind, and so contains minimum number of parameters, and only four free parameters. Moreover, all model parameters can be measured experimentally. Free parameters were tuned by fitting the model to experimental data from the literature, using B16-F10 murine melanoma implanted into wild type (C57BL/6), as well as into immunodeficient (NSG) mice strains, and treated with anti-PD-1 antibodies. The model's predictive ability was verified on two independent datasets from literature with different but well-known inputs. To identify possible biomarkers of response to anti-PD-1 immunotherapy, sensitivity study of key model parameters was performed. Good agreement between the simulated tumour growth curves and the experimental data was achieved, with mean relative deviations in the range of 13%-20%. Our sensitivity study demonstrated that major histocompatibility complex (MHC) class I expression was the only parameter able to clearly discriminate responders from non-responders to anti-PD-1 therapy. Additionally, the results of sensitivity studies suggest that MHC class I expression might affect the predictive ability of other biomarkers that are currently used in the clinics, such as PD-1 ligand (PD-L1) expression. Interestingly, our model predicts the best response to anti-PD-1 therapy for subjects with moderate PD-L1 values. Such computational models show promise to support, guide and accelerate future immunotherapy research.
Abstract
Immune checkpoint inhibitors have shown impressive benefits for patients with various types of cancer. However, patients who respond are still a minority. To improve the response rates, ...combinations of various immunotherapies, as well as combinations with other conventional therapies, have been extensively studied. Due to an unmanageably high number of all possible combinations and dosing regimens, alternatives to the costly and time-consuming trial-and-error approach are of utmost importance. Our main goal was to develop a verifiable computational model that would analyze the tumor response to anti-PD-1 antibodies and provide suggestions about the possible biomarkers of response to anti-PD-1 immunotherapy. Our model was built with validation in mind, and so contains minimum number of parameters. Moreover, all parameters can be measured experimentally. The model was tuned and validated in vivo using 3 murine tumor cell lines (B16-F10, CT26, 4T1) in 3 different settings: (1) growth of tumors in NUDE mice to assess intrinsic tumor growth in the absence of T-cells, (2) growth of tumors in wild-type (WT) mice to assess the effect of the immune system on untreated tumor growth, and (3) growth of tumors in WT mice receiving anti-PD-1 antibodies to assess the therapeutic effect. MHC Class I and PD-L1 expression on tumor cells was measured in vitro using flow cytometry to assess the parameters associated with immunogenicity of selected tumor cell lines. Single nucleotide variations (SNV) data, indicative of the mutational load, were taken from literature. Finally, we performed a sensitivity study of key model parameters to identify possible biomarkers of tumor response to anti-PD-1 therapy. In vitro results showed comparable PD-L1 expression in all 3 cell lines (11%-21%), while MHC class I expression varied significantly between B16-F10 (2.8%), 4T1 (99.5%), and CT26 (99.9%). Additionally, SNV data indicated an order of magnitude higher CT26 SNV (3023) compared to 4T1 (293), and more than 3 times higher compared to B16-F10 (908). Using the above-measured parameters our computational model was able to reproduce all in vivo experiments. The model suggests that the average occupancy of PD-1 receptors on tumor-infiltrating T cells by anti-PD-1 antibodies is much higher in CT26 (74%) compared to 4T1 (30%) or B16-F10 (8%). It indicates that the ability of antibodies to penetrate the tumor might vary depending on the tumor type. The results of the sensitivity study suggested that a combination of MHC class I, PD-L1 and SNV might be superior for predicting tumor response to anti-PD-1 compared to either of the biomarkers alone. Namely, CT26, the cell line with high MHC class I, high SNV and moderate PD-L1 expression, was the only cell line where complete responses to anti-PD-1 antibodies were observed experimentally. Despite simplified description of the reality, our model generates meaningful hypotheses to be tested in future (pre)clinical trials. Such models show promise to support, guide and accelerate immunotherapy research.
Citation Format: Damijan Valentinuzzi, Katja Ursic, Urban Simoncic, Matea Maruna, Marusa Turk, Martina Vrankar, Maja Cemazar, Gregor Sersa, Robert Jeraj. Computational modeling analysis of the tumor response to anti-PD-1 immunotherapy abstract. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A09.
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