Enhanced perfusion following exposure to radiotherapy: A theoretical investigation.

Tumour angiogenesis leads to the formation of blood vessels that are structurally and spatially heterogeneous. Poor blood perfusion, in conjunction with increased hypoxia and oxygen heterogeneity, impairs a tumour's response to radiotherapy. The optimal strategy for enhancing tumour perfusion remains unclear, preventing its regular deployment in combination therapies. In this work, we first identify vascular architectural features that correlate with enhanced perfusion following radiotherapy, using in vivo imaging data from vascular tumours. Then, we present a novel computational model to determine the relationship between these architectural features and blood perfusion in silico. If perfusion is defined to be the proportion of vessels that support blood flow, we find that vascular networks with small mean diameters and large numbers of angiogenic sprouts show the largest increases in perfusion post-irradiation for both biological and synthetic tumours. We also identify cases where perfusion increases due to the pruning of hypoperfused vessels, rather than blood being rerouted. These results indicate the importance of considering network composition when determining the optimal irradiation strategy. In the future, we aim to use our findings to identify tumours that are good candidates for perfusion enhancement and to improve the efficacy of combination therapies.

The influence of cytotoxic drugs on the immunophenotype of blast cells in paediatric B precursor acute lymphoblastic leukaemia.

BACKGROUND: Flow cytometry plays is important in the diagnosis of acute lymphoblastic leukaemia (ALL) and when antigen-specific immunotherapy is indicated. We have investigated the effects of prednisolone, vincristine, daunorubicin, asparaginase and methotrexate on the antigen expression on blast cells that could influence the planning of antigen-specific therapy as well as risk-based treatment assignment. PATIENTS AND METHODS: Patients aged ≤ 17 years with de novo B-cell ALL (B-ALL) were enrolled in the study. Blast cells were isolated and exposed in vitro to 5 individual cytotoxic drugs in logarithmically increasing concentrations. Then, the expression of CD10, CD19, CD20, CD27, CD34, CD45, CD58, CD66c and CD137 antigens was determined by quantitative flow cytometry. RESULTS: Cytotoxic drugs caused dose-dependent or dose-independent modulation of antigen expression. Daunorubicin caused a dose-dependent down-modulation of CD10, CD19, CD34, CD45 and CD58 and an up-modulation of CD137. Vincristine caused a dose-dependent down-modulation of CD19 and CD58 and an up-modulation of CD45. Daunorubicin also caused dose-independent down-modulation of CD27 and prednisolone down-modulation of CD10, CD19, CD27, CD34 and CD58. Down-modulation of CD20 was detected only in relation to the specific dose of daunorubicin. CONCLUSIONS: The results of the study have shown that cytotoxic drugs can alter the expression of antigens that are important for immunotherapy. Importantly, daunorubicin, prednisolone and vincristine caused down-modulation of CD19 and CD58, suggesting that these drugs are better avoided during bridging therapy prior to bispecific antibodies or CAR-T cell therapy. In addition, immunophenotypic changes on blast cells induced by different drugs could also influence risk-based treatment assignment.

Characterization of two distinct immortalized endothelial cell lines, EA.hy926 and HMEC-1, for in vitro studies: exploring the impact of calcium electroporation, Ca2+ signaling and transcriptomic profiles.

BACKGROUND: Disruption of Ca2+ homeostasis after calcium electroporation (CaEP) in tumors has been shown to elicit an enhanced antitumor effect with varying impacts on healthy tissue, such as endothelium. Therefore, our study aimed to determine differences in Ca2+ kinetics and gene expression involved in the regulation of Ca2+ signaling and homeostasis, as well as effects of CaEP on cytoskeleton and adherens junctions of the established endothelial cell lines EA.hy926 and HMEC-1. METHODS: CaEP was performed on EA.hy926 and HMEC-1 cells with increasing Ca2+ concentrations. Viability after CaEP was assessed using Presto Blue, while the effect on cytoskeleton and adherens junctions was evaluated via immunofluorescence staining (F-actin, α-tubulin, VE-cadherin). Differences in intracellular Ca2+ regulation ([Ca2+]i) were determined with spectrofluorometric measurements using Fura-2-AM, exposing cells to DPBS, ionomycin, thapsigargin, ATP, bradykinin, angiotensin II, acetylcholine, LaCl3, and GdCl3. Molecular distinctions were identified by analyzing differentially expressed genes and pathways related to the cytoskeleton and Ca2+ signaling through RNA sequencing. RESULTS: EA.hy926 cells, at increasing Ca2+ concentrations, displayed higher CaEP susceptibility and lower survival than HMEC-1. Immunofluorescence confirmed CaEP-induced, time- and Ca2+-dependent morphological changes in EA.hy926's actin filaments, microtubules, and cell-cell junctions. Spectrofluorometric Ca2+ kinetics showed higher amplitudes in Ca2+ responses in EA.hy926 exposed to buffer, G protein coupled receptor agonists, bradykinin, and angiotensin II compared to HMEC-1. HMEC-1 exhibited significantly higher [Ca2+]i changes after ionomycin exposure, while responses to thapsigargin, ATP, and acetylcholine were similar in both cell lines. ATP without extracellular Ca2+ ions induced a significantly higher [Ca2+]i rise in EA.hy926, suggesting purinergic ionotropic P2X and metabotropic P2Y receptor activation. RNA-sequencing analysis showed significant differences in cytoskeleton- and Ca2+-related gene expression, highlighting upregulation of ORAI2, TRPC1, TRPM2, CNGA3, TRPM6, and downregulation of TRPV4 and TRPC4 in EA.hy926 versus HMEC-1. Moreover, KEGG analysis showed upregulated Ca2+ import and downregulated export genes in EA.hy926. CONCLUSIONS: Our finding show that significant differences in CaEP response and [Ca2+]i regulation exist between EA.hy926 and HMEC-1, which may be attributed to distinct transcriptomic profiles. EA.hy926, compared to HMEC-1, displayed higher susceptibility and sensitivity to [Ca2+]i changes, which may be linked to overexpression of Ca2+-related genes and an inability to mitigate changes in [Ca2+]i. The study offers a bioinformatic basis for selecting EC models based on research objectives.

Partial-Volume Irradiation of Murine Tumors.

Radiotherapy is a widely used approach for cancer treatment. However, delivering a single high dose of radiation to bulky tumors can be challenging due to the toxicities induced in the surrounding healthy tissue. To overcome this issue, a nonuniform high dose can be delivered using partial-volume tumor irradiation or spatially fractionated radiotherapy (SFRT). Moreover, SFRT has the potential to induce a stronger antitumor immune response compared to traditional radiotherapy due to the preservation of immune cells in the unirradiated tumor regions. There are several SFRT approaches, including GRID therapy, three-dimensional GRID therapy (LATTICE), microbeam radiation therapy (MRT), and Stereotactic Body Radiation Therapy for PArtial Tumor irradiation targeting exclusively the HYpoxic segment (SBRT-PATHY). The following protocol describes partial-volume tumor irradiation, a technique that enables dose delivery to only a part of the tumor in mice using an X-ray generator and collimators of different dimensions that limit the size of the irradiation field.

Imaging of Extravasation of Splenocytes in the Dorsal Skinfold Window Chamber.

Infiltration of immune cells into the tumor is one of the major drivers of antitumor immune response, which can direct the outcome of anticancer therapies. In mice, implantation of dorsal skinfold window chamber (DSWC) combined with intravital confocal fluorescence microscopy allows real-time observation of splenocyte extravasation and infiltration into tumors. Here, we describe a detailed procedure of the DSWC implantation, splenocyte isolation and fluorescent labeling, intravenous injection of labeled splenocytes, and imaging of splenocyte extravasation into tumors using confocal fluorescence microscopy.

What We Learned about the Feasibility of Gene Electrotransfer for Vaccination on a Model of COVID-19 Vaccine.

DNA vaccination is one of the emerging approaches for a wide range of applications, including prophylactic vaccination against infectious diseases and therapeutic vaccination against cancer. The aim of this study was to evaluate the feasibility of our previously optimized protocols for gene electrotransfer (GET)-mediated delivery of plasmid DNA into skin and muscle tissues on a model of COVID-19 vaccine. Plasmids encoding the SARS-CoV-2 proteins spike (S) and nucleocapsid (N) were used as the antigen source, and a plasmid encoding interleukin 12 (IL-12) was used as an adjuvant. Vaccination was performed in the skin or muscle tissue of C57BL/6J mice on days 0 and 14 (boost). Two weeks after the boost, blood, spleen, and transfected tissues were collected to determine the expression of S, N, IL-12, serum interferon-γ, the induction of antigen-specific IgG antibodies, and cytotoxic T-cells. In accordance with prior in vitro experiments that indicated problems with proper expression of the S protein, vaccination with S did not induce S-specific antibodies, whereas significant induction of N-specific antibodies was detected after vaccination with N. Intramuscular vaccination outperformed skin vaccination and resulted in significant induction of humoral and cell-mediated immunity. Moreover, both boost and adjuvant were found to be redundant for the induction of an immune response. Overall, the study confirmed the feasibility of the GET for DNA vaccination and provided valuable insights into this approach.

The effectiveness of calcium electroporation combined with gene electrotransfer of a plasmid encoding IL-12 is tumor type-dependent.

Introduction: In calcium electroporation (CaEP), electroporation enables the cellular uptake of supraphysiological concentrations of Ca2+, 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. Methods: The effect of CaEP was tested in vitro as well as in vivo 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. Results: In vitro, 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 in vivo; 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. Conclusions: Mice bearing 4T1 tumors responded better to CaEP in vivo than mice bearing B16-F10 tumors, even though a similar response was observed in vitro. 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.

Effect of electrochemotherapy on myogenesis of mouse C2C12 cells in vitro.

Electrochemotherapy (ECT) is a local ablative therapy for the treatment of different skin and subcutaneous tumors and certain tumors in internal organs. Skeletal muscle represents a major tumor- surrounding tissue, exposed to side effects of ECT. At the cellular level, side-effects of ECT on skeletal muscle and underlying mechanisms have not been examined yet. Thus, we aimed to determine the effect of ECT in the mouse muscle cell line C2C12 during in vitro myogenesis. We evaluated the electroporation efficiency and viability of C2C12 myotubes at increasing voltages (200-1300 V/cm) using propidium iodide (PI). Permeabilization of PI into the cells was voltage-dependent accounting up to 97 % efficiency at the highest voltage. High cell viability and myotube integrity were maintained until 4 days after electroporation. ECT with the cytostatic drugs bleomycin and cisplatin decreased the viability of C2C12 myoblasts and myotubes in a dose-dependent manner. However, myoblasts were more sensitive to ECT than myotubes. Increased secretion of IL-6, observed 3 days after ECT, confirming its effects on early myogenesis. Only minor effects of ECT were observed in treated myotubes. These results contribute to the safety profile of ECT in tumor treatment.

HPV-positive murine oral squamous cell carcinoma: development and characterization of a new mouse tumor model for immunological studies.

BACKGROUND: Infection with high-risk human papillomavirus (HPV) strains is one of the risk factors for the development of oral squamous cell carcinoma (OSCC). Some patients with HPV-positive OSCC have a better prognosis and respond better to various treatment modalities, including radiotherapy or immunotherapy. However, since HPV can only infect human cells, there are only a few immunocompetent mouse models available that enable immunological studies. Therefore, the aim of our study was to develop a transplantable immunocompetent mouse model of HPV-positive OSCC and characterize it in vitro and in vivo. METHODS: Two monoclonal HPV-positive OSCC mouse cell lines were established by inducing the expression of HPV-16 oncogenes E6 and E7 in the MOC1 OSCC cell line using retroviral transduction. After confirming stable expression of HPV-16 E6 and E7 with quantitative real-time PCR and immunofluorescence staining, the cell lines were further characterized in vitro using proliferation assay, wound healing assay, clonogenic assay and RNA sequencing. In addition, tumor models were characterized in vivo in C57Bl/6NCrl mice in terms of their histological properties, tumor growth kinetics, and radiosensitivity. Furthermore, immunofluorescence staining of blood vessels, hypoxic areas, proliferating cells and immune cells was performed to characterize the tumor microenvironment of all three tumor models. RESULTS: Characterization of the resulting MOC1-HPV cell lines and tumor models confirmed stable expression of HPV-16 oncogenes and differences in cell morphology, in vitro migration capacity, and tumor microenvironment characteristics. Although the cell lines did not differ in their intrinsic radiosensitivity, one of the HPV-positive tumor models, MOC1-HPV K1, showed a significantly longer growth delay after irradiation with a single dose of 15 Gy compared to parental MOC1 tumors. Consistent with this, MOC1-HPV K1 tumors had a lower percentage of hypoxic tumor area and a higher percentage of proliferating cells. Characteristics of the newly developed HPV-positive OSCC tumor models correlate with the transcriptomic profile of MOC1-HPV cell lines. CONCLUSIONS: In conclusion, we developed and characterized a novel immunocompetent mouse model of HPV-positive OSCC that exhibits increased radiosensitivity and enables studies of immune-based treatment approaches in HPV-positive OSCC.

Effects of Electrochemotherapy on Immunologically Important Modifications in Tumor Cells.

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.

Estimating quantitative physiological and morphological tissue parameters of murine tumor models using hyperspectral imaging and optical profilometry.

Understanding tumors and their microenvironment are essential for successful and accurate disease diagnosis. Tissue physiology and morphology are altered in tumors compared to healthy tissues, and there is a need to monitor tumors and their surrounding tissues, including blood vessels, non-invasively. This preliminary study utilizes a multimodal optical imaging system combining hyperspectral imaging (HSI) and three-dimensional (3D) optical profilometry (OP) to capture hyperspectral images and surface shapes of subcutaneously grown murine tumor models. Hyperspectral images are corrected with 3D OP data and analyzed using the inverse-adding doubling (IAD) method to extract tissue properties such as melanin volume fraction and oxygenation. Blood vessels are segmented using the B-COSFIRE algorithm from oxygenation maps. From 3D OP data, tumor volumes are calculated and compared to manual measurements using a vernier caliper. Results show that tumors can be distinguished from healthy tissue based on most extracted tissue parameters ( p < 0.05 ). Furthermore, blood oxygenation is 50% higher within the blood vessels than in the surrounding tissue, and tumor volumes calculated using 3D OP agree within 26% with manual measurements using a vernier caliper. Results suggest that combining HSI and OP could provide relevant quantitative information about tumors and improve the disease diagnosis.

Design, Development, and Testing of a Device for Gene Electrotransfer to Skin Cells In Vivo.

Gene electrotransfer (GET) is considered one of the most efficient, safe, reproducible, and cost-effective methods of gene therapy, in which a gene is delivered to the cells in the form of a plasmid DNA vector by a method known as electroporation. To achieve successful electroporation, cells must be exposed to sufficiently high electric fields generated by short-duration, high-voltage electrical pulses that result in a temporary increase in plasma membrane permeability. The electrical pulses are generated by pulse generators (electroporators) and delivered to the cells via electrodes (applicators). However, there is a lack of standardized pulse delivery protocols as well as certified clinical pulse generators and applicators for gene delivery. In this paper, the development of a new pulse generator, applicator, and pulse delivery protocol for GET to skin cells is presented. A numerical model of electroporated skin developed and tested for two electrode configurations and two different pulse delivery protocols is also presented. An alternative pulse delivery protocol was proposed. The developed pulse generator, applicator, and the proposed pulse delivery protocol were then used in vivo for GET to skin cells in mice. The results showed high efficiency of the proposed pulse delivery protocol for the purpose of GET in mouse skin cells. Specifically, electroporation with the developed pulse generator, applicator, and proposed pulse delivery protocol resulted in higher gene expression in skin cells compared to the currently used pulse generator, applicator, and pulse delivery protocol.

Endothelial cell death after ionizing radiation does not impair vascular structure in mouse tumor models.

The effect of radiation therapy on tumor vasculature has long been a subject of debate. Increased oxygenation and perfusion have been documented during radiation therapy. Conversely, apoptosis of endothelial cells in irradiated tumors has been proposed as a major contributor to tumor control. To examine these contradictions, we use multiphoton microscopy in two murine tumor models: MC38, a highly vascularized, and B16F10, a moderately vascularized model, grown in transgenic mice with tdTomato-labeled endothelium before and after a single (15 Gy) or fractionated (5 × 3 Gy) dose of radiation. Unexpectedly, even these high doses lead to little structural change of the perfused vasculature. Conversely, non-perfused vessels and blind ends are substantially impaired after radiation accompanied by apoptosis and reduced proliferation of their endothelium. RNAseq analysis of tumor endothelial cells confirms the modification of gene expression in apoptotic and cell cycle regulation pathways after irradiation. Therefore, we conclude that apoptosis of tumor endothelial cells after radiation does not impair vascular structure.

Multiscale topology characterizes dynamic tumor vascular networks.

Advances in imaging techniques enable high-resolution three-dimensional (3D) visualization of vascular networks over time and reveal abnormal structural features such as twists and loops, and their quantification is an active area of research. Here, we showcase how topological data analysis, the mathematical field that studies the "shape" of data, can characterize the geometric, spatial, and temporal organization of vascular networks. We propose two topological lenses to study vasculature, which capture inherent multiscale features and vessel connectivity, and surpass the single-scale analysis of existing methods. We analyze images collected using intravital and ultramicroscopy modalities and quantify spatiotemporal variation of twists, loops, and avascular regions (voids) in 3D vascular networks. This topological approach validates and quantifies known qualitative trends such as dynamic changes in tortuosity and loops in response to antibodies that modulate vessel sprouting; furthermore, it quantifies the effect of radiotherapy on vessel architecture.

Treatment of skin tumors with intratumoral interleukin 12 gene electrotransfer in the head and neck region: a first-in-human clinical trial protocol.

BACKGROUND: Immune therapies are currently under intensive investigation providing in many cases excellent responses in different tumors. Other possible approach for immunotherapy is a targeted intratumoral delivery of interleukin 12 (IL-12), a cytokine with anti-tumor effectiveness. Due to its immunomodulatory action, it can be used as an imunostimulating component to in situ vaccinating effect of local ablative therapies. We have developed a phIL12 plasmid devoid of antibiotic resistance marker with a transgene for human IL-12 p70 protein. The plasmid can be delivered intratumorally by gene electrotransfer (GET). PATIENTS AND METHODS: Here we present a first-in-human clinical trial protocol for phIL12 GET (ISRCTN15479959, ClinicalTrials NCT05077033). The study is aimed at evaluating the safety and tolerability of phIL12 GET in treatment of basal cell carcinomas in patients with operable tumors in the head and neck region. The study is designed as an exploratory, dose escalating study with the aim to determine the safety and tolerability of the treatment and to identify the dose of plasmid phIL12 that is safe and elicits its biological activity. CONCLUSIONS: The results of this trail protocol will therefore provide the basis for the use of phIL12 GET as an adjuvant treatment to local ablative therapies, to potentially increase their local and elicit a systemic response.

Sunitinib potentiates the cytotoxic effect of electrochemotherapy in pancreatic carcinoma cells.

BACKGROUND: One of the new treatment options for unresectable locally advanced pancreatic cancer is electrochemotherapy (ECT), a local ablative therapy that potentiates the entry of chemotherapeutic drugs into the cells, by the application of an electric field to the tumor. Its feasibility and safety were demonstrated in preclinical and clinical studies; however, there is a lack of preclinical studies assessing the actions of different drugs used in ECT, their mechanisms and interactions with other target drugs that are used in clinical practice. MATERIALS AND METHODS: The aim of the study was to determine the cytotoxicity of two chemotherapeutic drugs usually used in ECT (bleomycin and cisplatin) in the BxPC-3 human pancreatic carcinoma cell line and evaluate the interactions of ECT with the targeted drug sunitinib. First, the cytotoxicity of ECT using both chemotherapeutics was determined. In the next part, the interactions of ECT and sunitinib were evaluated through determination of combined cytotoxicity, sunitinib targets and kinetics of cell death. RESULTS: The results demonstrate that ECT is effective in pancreatic cancer cell line, especially when bleomycin is used, with the onset of cell death in the first hours after the treatment, reaching a plateau at 20 hours after the treatment. Furthermore, we provide the rationale for combining ECT with bleomycin and the targeted drug sunitinib to potentiate cytotoxicity. The combined treatment of sunitinib and ECT was synergistic for bleomycin only at the highest used concentration of bleomycin 0.14 µM, whereas with lower doses of bleomycin, this effect was not observed. The interaction of ECT and treatment with sunitinib was confirmed by course of the cell death, also indicating on synergism. CONCLUSIONS: ECT and sunitinib combined treatment has clinical potential, and further studies are warranted.

Non-Clinical In Vitro Evaluation of Antibiotic Resistance Gene-Free Plasmids Encoding Human or Murine IL-12 Intended for First-in-Human Clinical Study.

Interleukin 12 (IL-12) is a key cytokine that mediates antitumor activity of immune cells. To fulfill its clinical potential, the development is focused on localized delivery systems, such as gene electrotransfer, which can provide localized delivery of IL-12 to the tumor microenvironment. Gene electrotransfer of the plasmid encoding human IL-12 is already in clinical trials in USA, demonstrating positive results in the treatment of melanoma patients. To comply with EU regulatory requirements for clinical application, which recommend the use of antibiotic resistance gene-free plasmids, we constructed and developed the production process for the clinical grade quality antibiotic resistance gene-free plasmid encoding human IL-12 (p21-hIL-12-ORT) and its ortholog encoding murine IL-12 (p21-mIL-12-ORT). To demonstrate the suitability of the p21-hIL-12-ORT or p21-mIL-12-ORT plasmid for the first-in-human clinical trial, the biological activity of the expressed transgene, its level of expression and plasmid copy number were determined in vitro in the human squamous cell carcinoma cell line FaDu and the murine colon carcinoma cell line CT26. The results of the non-clinical evaluation in vitro set the basis for further in vivo testing and evaluation of antitumor activity of therapeutic molecules in murine models as well as provide crucial data for further clinical trials of the constructed antibiotic resistance gene-free plasmid in humans.

Mutational burden, MHC-I expression and immune infiltration as limiting factors for in situ vaccination by TNFα and IL-12 gene electrotransfer.

In situ vaccination is a promising immunotherapeutic approach, where various local ablative therapies are used to induce an immune response against tumor antigens that are released from the therapy-killed tumor cells. We recently proposed using intratumoral gene electrotransfer for concomitant transfection of a cytotoxic cytokine tumor necrosis factor-α (TNFα) to induce in situ vaccination, and an immunostimulatory cytokine interleukin 12 (IL-12) to boost the primed immune response. Here, our aim was to test the local and systemic effectiveness of the approach in tree syngeneic mouse tumor models and associate it with tumor immune profiles, characterized by tumor mutational burden, immune infiltration and expression of PD-L1 and MHC-I on tumor cells. While none of the tested characteristic proved predictive for local effectiveness, high tumor mutational burden, immune infiltration and MHC-I expression were associated with higher abscopal effectiveness. Hence, we have confirmed that both the abundance and presentation of tumor antigens as well as the absence of immunosuppressive mechanisms are important for effective in situ vaccination. These findings provide important indications for future development of in situ vaccination based treatments, and for the selection of tumor types that will most likely benefit from it.

PARP inhibitor olaparib has a potential to increase the effectiveness of electrochemotherapy in BRCA1 mutated breast cancer in mice.

Electrochemotherapy (ECT), a local therapy, has different effectiveness among tumor types. In breast cancer, its effectiveness is low; therefore, combined therapies are needed. The aim of our study was to combine ECT with PARP inhibitor olaparib, which could inhibit the repair of bleomycin or cisplatin induced DNA damage and potentiate the effectiveness of ECT. The effects of combined therapy were studied in BRCA1 mutated (HCC1937) and non-mutated (HCC1143) triple negative breast cancer cell lines. Therapeutic effectiveness was studied in 2D and 3D cell cultures and in vivo on subcutaneous HCC1937 tumor model in mice. The underlying mechanism of combined therapy was determined with the evaluation of γH2AX foci. Combined therapy of ECT with bleomycin and olaparib potentiated the effectiveness of ECT in BRCA1 mutated HCC1937, but not in non-mutated HCC1143 cells. The combined therapy had a synergistic effect, which was due to the increased number of DNA double strand breaks. Addition of olaparib to ECT with bleomycin in vivo in HCC1937 tumor model had only minimal effect, indicating repetitive olaparib treatment would be needed. This study demonstrates that DNA repair inhibiting drugs, like olaparib, have the potential to increase the effectiveness of ECT with bleomycin.