Differential association of CD68+ and CD163+ macrophages with macrophage enzymes, whole tumour gene expression and overall survival in advanced melanoma.

BACKGROUND: The density and phenotype of tumour-associated macrophages have been linked with prognosis in a range of solid tumours. While there is strong preclinical evidence that tumour-associated macrophages promote aspects of tumour progression, it can be challenging to infer clinical activity from surface markers and ex vivo behaviour. We investigated the association of macrophage infiltration with prognosis and functional changes in the tumour microenvironment in primary human melanoma. METHODS: Fifty-seven formalin-fixed, paraffin-embedded primary melanomas were analysed by immunohistochemical analysis of CD68, CD163, inducible nitric oxide synthase (iNOS) and arginase expression. RNA sequencing was performed on serial sections of 20 of the stained tumours to determine the influence of macrophage infiltration on gene expression. RESULTS: CD68+ cells are a functionally active subset of macrophages that are associated with increased iNOS and arginase staining and altered gene expression. In comparison, while there is a greater accumulation of CD163+ macrophages in larger tumours, these cells are comparatively inactive, with no association with the level of iNOS or arginase staining, and no effect on gene expression within the tumour. The infiltration of either subset of macrophages did not correlate to overall survival. CONCLUSIONS: Thus, melanomas contain distinct macrophage populations with diverse phenotypes, but with no observable prognostic role.

Melanoma-conditioned medium promotes cytotoxic immune responses by murine bone marrow-derived monocytes despite their expression of 'M2' markers.

Macrophages have been shown to infiltrate a wide range of malignancies and are often considered to promote tumour survival, growth and spread. However, the source and behaviour of discrete tumour-associated macrophage populations are still poorly understood. Here we show a novel method for the rational development of bone marrow-derived monocytes appropriate for the study of processes which involve the contribution of circulating inflammatory monocytes. We have shown that in response to tumour-conditioned medium, these cells upregulate CD206 and CD115, markers traditionally associated with M2-type macrophages. Treated cells show reduced capacity for cytokine secretion but significantly impact CD4+ and CD8+ T-cell proliferation and polarization. Coculture with conditioned bone marrow-derived monocytes significantly reduced CD4+ T-cell proliferation but increased CD8+ T-cell proliferation and granzyme B expression with significant induction of IFNγ secretion by both CD4+ and CD8+ T cells, indicating that these cells may have a role in promoting anti-cancer immunity.

Glycolysis inhibition as a cancer treatment and its role in an anti-tumour immune response.

Increased glycolysis is the main source of energy supply in cancer cells that use this metabolic pathway for ATP generation. Altered energy metabolism is a biochemical fingerprint of cancer cells that represents one of the "hallmarks of cancer". The immune system can prevent tumour growth by eliminating cancer cells but this editing process ultimately results in poorly immunogenic cells remaining allowing for unchallenged tumour growth. In this review we look at the glycolysis pathway as a target for cancer treatments. We also examine the interplay between the glycolysis modulation and the immune response as an anti-cancer therapy.

Clinical evaluation of macrophages in cancer: role in treatment, modulation and challenges.

The focus of immunotherapeutics has been placed firmly on anti-tumour T cell responses. Significant progress has been made in the treatment of both local and systemic malignancies, but low response rates and rising toxicities are limiting this approach. Advancements in the understanding of tumour immunology are opening up a new range of therapeutic targets, including immunosuppressive factors in the tumour microenvironment. Macrophages are a heterogeneous group of cells that have roles in innate and adaptive immunity and tissue repair, but become co-opted by tumours to support tumour growth, survival, metastasis and immunosuppression. Macrophages also support tumour resistance to conventional therapy. In preclinical models, interference with macrophage migration, macrophage depletion and macrophage re-education have all been shown to reduce tumour growth and support anti-tumour immune responses. Here we discuss the role of macrophages in prognosis and sensitivity to therapy, while examining the significant progress which has been made in modulating the behaviour of these cells in cancer patients.

Effective treatment of intractable cutaneous metastases of breast cancer with electrochemotherapy: Ten-year audit of single centre experience.

PURPOSE: Electrochemotherapy (ECT) is the application of electric pulses to tumour tissue to render the cell membranes permeable to usually impermeant hydrophilic anti-cancer drugs, thereby enhancing cytotoxic effects. We sought to ascertain whether ECT can be an effective palliative treatment for cutaneous metastases of breast cancer. METHODS: This work reports data from the European Standard Operating Procedures for Electrochemotherapy trial (EudraCT Number: 2004-002183-18). In combination with systemic and/or intratumoural bleomycin, optimised electric pulses were delivered to locally recurrent or metastatic cutaneous breast cancer lesions. Follow-up continued until December 2014. RESULTS: Between February 2004 and December 2014, twenty-four patients were treated. All patients had received prior multimodal therapy. In total, the patient cohort had, or developed, 242 lesions. Two hundred and 36 lesions were treated, with 34 lost to follow-up. An objective response was seen in 161 of 202 lesions (79.7%), with a complete response observed in 130 (64.3%). Thirty-nine lesions (19.3%) did not respond, while 2 (1%) progressed following ECT. 17 (73.9%) patients received two or fewer treatments. A minimum of a partial response was seen in at least 50% of treated lesions in 18 of the 24 (75%) patients. Smaller lesions were more likely to have an objective response (Chi-square test for trend, p < 0.001). CONCLUSIONS: Electrochemotherapy is an effective treatment for cutaneous breast cancer lesions that have proven refractory to standard therapies. As smaller lesions were found to be more responsive, we suggest that ECT should be considered as an early treatment modality, within multimodal treatment strategies.

Antitumour responses induced by a cell-based Reovirus vaccine in murine lung and melanoma models.

BACKGROUND: The ever increasing knowledge in the areas of cell biology, the immune system and the mechanisms of cancer are allowing a new phase of immunotherapy to develop. The aim of cancer vaccination is to activate the host immune system and some success has been observed particularly in the use of the BCG vaccine for bladder cancer as an immunostimulant. Reovirus, an orphan virus, has proven itself as an oncolytic virus in vitro and in vivo. Over 80 % of tumour cell lines have been found to be susceptible to Reovirus infection and it is currently in phase III clinical trials. It has been shown to induce immune responses to tumours with very low toxicities. METHODS: In this study, Reovirus was examined in two main approaches in vivo, in mice, using the melanoma B16F10 and Lewis Lung Carcinoma (LLC) models. Initially, mice were treated intratumourally (IT) with Reovirus and the immune responses determined by cytokine analysis. Mice were also vaccinated using a cell-based Reovirus vaccine and subsequently exposed to a tumourigenic dose of cells (B16F10 or LLC). Using the same cell-based Reovirus vaccine, established tumours were treated and subsequent immune responses and virus retrieval investigated. RESULTS: Upregulation of several cytokines was observed following treatment and replication-competent virus was also retrieved from treated tumours. Varying levels of cytokine upregulation were observed and no replication-competent virus was retrieved in vaccine-treated mice. Prolongation of survival and delayed tumour growth were observed in all models and an immune response to Reovirus, either using Reovirus alone or a cell-based vaccine was also observed in all mice. CONCLUSION: This study provides evidence of immune response to tumours using a cell-based Reovirus vaccine in both tumour models investigated, B16F10 and LLC, cytokine induction was observed with prolongation of survival in almost all cases which may suggest a new method for using Reovirus in the clinic.

Preclinical evaluation of an endoscopic electroporation system.

BACKGROUND AND STUDY AIMS: Targeted delivery of specific chemotherapeutic drugs into tumors can be achieved by delivering electrical pulses directly to the tumor tissue. This causes a transient formation of pores in the cell membrane that enables passive diffusion of normally impermeant drugs. A novel device has been developed to enable the endoscopic delivery of this tumor permeabilizing treatment. The aim of the preclinical studies described here was to investigate the efficacy and safety of this nonthermal ablation system in the treatment of gastrointestinal cancer models. METHODS: Murine, porcine, and canine gastrointestinal tumors and tissues were used to assess the efficacy and safety of electroporation delivered through the special device in combination with bleomycin. Tumor cell death, volume, and overall survival were recorded. RESULTS: Murine tumors treated with electrochemotherapy showed excellent responses, with cell death being induced rapidly, mainly via an apoptotic-type mechanism. Use of the system in canine gastrointestinal cancers demonstrated successful local endoluminal tumor resolution, with no safety or adverse effects noted. CONCLUSIONS: Electroporation via the new device in combination with bleomycin offers a nonthermal tumor ablative approach, and presents clinicians with a new option for the management of gastrointestinal cancers.

Local tumour ablative therapies: opportunities for maximising immune engagement and activation.

The relationship between cancer and the immune system is a complex one. The immune system can prevent tumour growth by eliminating cancer cells but this editing process ultimately results in poorly immunogenic cells remaining allowing for unchallenged tumour growth. In light of this, the focus of cancer treatment should be to maximise cancer elimination and the prevention of escape mechanisms. In this review we will examine current and emerging ablative treatment modalities that induce Immunogenic Cell Death (ICD), a special type of cell death that allows for immune cell involvement and the generation of an anti-tumour specific immune response. When paired with immune modulating agents, capable of potentiating the immune response and reversing the immune-suppressive environment created by tumours, we may be looking at the future of anti-cancer therapy.

Defining optimal parameters to maximize the effect of electrochemotherapy on lung cancer cells whilst preserving the integrity of immune cells.

Electrochemotherapy (ECT) is becoming an established therapy for melanoma and is under investigation for application in additional cancer types. One potential cancer type that may benefit from ECT is lung cancer as lung cancer treatments remain unable to deliver long-lasting treatment responses. Given the importance of the immune system in lung cancer, here we have also examined the impact of ECT on immune populations. The impact of electroporation and ECT on three human lung cancer cell lines (A549, H460, SK-MES 1), one murine cell line (LLC) and murine T cells, dendritic cells and macrophages was examined. The viability, metabolic activity and recovery potential post-treatment of all cell types was determined to evaluate the potential utility of ECT as a lung cancer treatment. Our findings demonstrate that cisplatin at 11 µM would be the suggested drug of choice when using ECT for lung cancer treatment. Our study also shows that T cells are not impacted by any tested condition, whilst dendritic cells and macrophages are significantly negatively impacted by electric field strengths surpassing 800 V/cm in vitro. Therefore, current ECT protocols (using 1000 V/cm in vivo) might need to adapted to improve viability of the immune population, thus improving therapy outcomes.

Electrochemotherapy and immune interactions; A boost to the system?

Electrochemotherapy (ECT), the application of an electric impulse to deliver chemotherapy drugs into cells, has been in clinical trials since the early 1990s and has been used for a variety of different malignancies including melanoma and sarcoma. A standard operating procedure for the use of ECT in clinical settings has been established since 2006. ECT is very effective in reducing the local tumour burden via T-cell dependent killing of the cancer cells; however abscopal effects are not consistently observed. Currently little is known or understood about how ECT affects the immune cell population within the treated tumour and how these changes could impact the immune response. In this manuscript, we will review the current knowledge on ECT in the context of its interactions with the immune system and discuss how the gained knowledge could be harnessed to develop a potent ECT-immune co-treatment combination (Electroimmunotherapy).

Electroporation of suspension cell lines - A proposed assay set for optimizations.

To make in vitro single cell electroporation protocols more comparable between various cancer types and groups, we propose a set of assays to test a range of electric field strengths at the start of any new project to determine the optimal electric field strength for a given cell line. While testing a range of electric field strengths, we kept the other ESOPE parameters constant (8 pulses, 100 µs pulse duration, 1 Hz pulse frequency). Basic assays were employed to measure short-term viability, effectiveness of treatment, metabolic activity, and recovery potential post-treatment to determine the optimal field strength for a particular cell line. Six cancer cell lines were tested, three of human (A549, A375 and Pan02) and three murine (LLC, B16F10 and MIA-PACA2). Our findings demonstrate that the optimal electroporation setting while keeping with all other ESOPE parameters are 800 V/cm for A549 and Pan02, 700 V/cm for A375, Mia-PACA2, and B16F10, and 1300 V/cm for LLC. Having an agreed upon set of assays to determine each cell lines optimal electric field strength should allow an improve translation of findings between cell lines for in vitro work from various groups and potentially improve translation into the clinic.

Testes and brain gene expression in precocious male and adult maturing Atlantic salmon (Salmo salar).

BACKGROUND: The male Atlantic salmon generally matures in fresh water upon returning after one or several years at sea. Some fast-growing male parr develop an alternative life strategy where they sexually mature before migrating to the oceans. These so called 'precocious' parr or 'sneakers' can successfully fertilise adult female eggs and so perpetuate their line. We have used a custom-built cDNA microarray to investigate gene expression changes occurring in the salmon gonad and brain associated with precocious maturation. The microarray has been populated with genes selected specifically for involvement in sexual maturation (precocious and adult) and in the parr-smolt transformation. RESULTS: Immature and mature parr collected from a hatchery-reared stock in January were significantly different in weight, length and condition factor. Changes in brain expression were small - never more than 2-fold on the microarray, and down-regulation of genes was much more pronounced than up-regulation. Significantly changing genes included isotocin, vasotocin, cathepsin D, anamorsin and apolipoprotein E. Much greater changes in expression were seen in the testes. Among those genes in the testis with the most significant changes in expression were anti-Mullerian hormone, collagen 1A, and zinc finger protein (Zic1), which were down-regulated in precocity and apolipoproteins E and C-1, lipoprotein lipase and anti-leukoproteinase precursor which were up-regulated in precocity. Expression changes of several genes were confirmed in individual fish by quantitative PCR and several genes (anti-Mullerian hormone, collagen 1A, beta-globin and guanine nucleotide binding protein (G protein) beta polypeptide 2-like 1 (GNB2L1) were also examined in adult maturing testes. Down-regulation of anti-Mullerian hormone was judged to be greater than 160-fold for precocious males and greater than 230-fold for November adult testes in comparison to July testes by this method. For anti-Mullerian hormone and guanine nucleotide binding protein beta polypeptide 2-like 1 expression changes in precocious males mirrored mature adults (November) but for collagen 1A and beta-globin the pattern was more complex. CONCLUSIONS: Expression changes in the fish brain during the process of precocious sexual maturation were small compared to those in the testes. Microarray analysis suggested down-regulation of housekeeping functions and up-regulation of a small number of specific processes. Transcriptional changes in the testes were much more pronounced with anti-Mullerian hormone playing a major role. Expression profiles for mature parr and maturing adult testes indicate subtle differences in gene expression between these two related groups.

The effect of calcium electroporation on viability, phenotype and function of melanoma conditioned macrophages.

Electroporation in combination with chemotherapy is an established treatment used on solid malignancies that results in enhanced chemotherapeutic uptake. Recent advances have begun to transition to the use of non-toxic compounds, such as calcium, in lieu of chemotherapy, which can also induce tumour cell death. While the effect of treatment on tumour cell death has been well characterized and has been shown to induce an immunogenic form of cell death, the effect of treatment on intratumoural immune cells has not been investigated. Here we present data showing the effect of calcium electroporation on immune cells, using melanoma-conditioned bone marrow-derived macrophages. Similar to tumour cells, macrophage cell membranes are susceptible to poration following treatment and subsequently reseal. Macrophages are less susceptible to calcium electroporation induced cell death in comparison to B16F10 melanoma cells. However treatment with electroporation with or without bleomycin or calcium was shown to affect macrophage phenotype and function. Coculture of calcium electroporated macrophages revealed that both the capacity of macrophages to stimulate and direct T cell responses are affected following exposure to treatment. We conclude that calcium electroporation has the potential to boost the immunogenic capacity of exposed tumour associated macrophages, and further research is warranted to determine if calcium electroporation can be optimised to generate systemic anti-cancer immune responses.

The Pentose Phosphate Pathway Dynamics in Cancer and Its Dependency on Intracellular pH.

The Pentose Phosphate Pathway (PPP) is one of the key metabolic pathways occurring in living cells to produce energy and maintain cellular homeostasis. Cancer cells have higher cytoplasmic utilization of glucose (glycolysis), even in the presence of oxygen; this is known as the "Warburg Effect". However, cytoplasmic glucose utilization can also occur in cancer through the PPP. This pathway contributes to cancer cells by operating in many different ways: (i) as a defense mechanism via the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to prevent apoptosis, (ii) as a provision for the maintenance of energy by intermediate glycolysis, (iii) by increasing genomic material to the cellular pool of nucleic acid bases, (iv) by promoting survival through increasing glycolysis, and so increasing acid production, and (v) by inducing cellular proliferation by the synthesis of nucleic acid, fatty acid, and amino acid. Each step of the PPP can be upregulated in some types of cancer but not in others. An interesting aspect of this metabolic pathway is the shared regulation of the glycolytic and PPP pathways by intracellular pH (pHi). Indeed, as with glycolysis, the optimum activity of the enzymes driving the PPP occurs at an alkaline pHi, which is compatible with the cytoplasmic pH of cancer cells. Here, we outline each step of the PPP and discuss its possible correlation with cancer.

Electrochemotherapy with cisplatin increases survival and induces immunogenic responses in murine models of lung cancer and colorectal cancer.

Electrochemotherapy is an emerging treatment modality for cancer patients which can effectively reduce tumour burden and induce immunogenic cell death. Electrochemotherapy is most commonly used with bleomycin as the drug of choice, here we examine the efficacy of electrochemotherapy with cisplatin. Electrochemotherapy with cisplatin was found to effectively reduce tumour growth in a range of murine models and induce significant intratumoural recruitment of myeloid and humoral immune cells. Following the observations of immune system mobilisation, we have shown an ability of electrochemotherapy to reduce metastatic potential as determined by tumour burden in the lung, and to exert an abscopal effect by reducing growth at distal untreated secondary tumours.

Electrochemotherapy Causes Caspase-Independent Necrotic-Like Death in Pancreatic Cancer Cells.

Pancreatic cancer represents a major challenge in oncology. Poor permeability of the pancreas and resistance to currently available therapies are impediments to improved patient survival. By transiently increasing cell membrane porosity and increasing drug uptake, Electrochemotherapy (ECT) has the potential to overcome these issues. In this study, we have evaluated the response of human and murine pancreatic cancer cells, in vitro, to electroporation in combination with Bleomycin, Cisplatin, or Oxaliplatin (ECT). The cytotoxic actions of all three drugs are potentiated when combined with electroporation in these cells. The biochemical and morphological changes post ECT are associated with immunogenic cell death that occurs with necroptosis rather than apoptosis. Moreover, ECT-induced cell death is rescued by Nec-1 suggesting that necroptosis may play a role in cell death mediated by cancer therapies.

Combination of electroporation delivered metabolic modulators with low-dose chemotherapy in osteosarcoma.

BACKGROUND: Osteosarcoma accounts for roughly 60% of all malignant bone tumors in children and young adults. The five-year survival rate for localized tumors after surgery and chemotherapy is approximately 70% whilst it drastically reduces to 15-30% in metastatic cases. Metabolic modulation is known to increase sensitivity of cancers to chemotherapy. A novel treatment strategy in Osteosarcoma is needed to battle this devastating malady. RESULTS: Electroporation-delivered metabolic modulators were more effective in halting the cell cycle of Osteosarcoma cells and this negatively affects their ability to recover and proliferate, as shown in colony formation assays. Electroporation-delivered metabolic modulators increase the sensitivity of Osteosarcoma cells to chemotherapy and this combination reduces their survivability. CONCLUSION: This novel treatment approach highlights the efficacy of electroporation in the delivery of metabolic modulators in Osteosarcoma cells, and increased sensitivity to chemotherapy allowing for a lower dose to be therapeutic. METHODS: Metabolic modulations of two Osteosarcoma cell lines were performed with clinically available modulators delivered using electroporation, and its combination with low-dose Cisplatin. The effects of Dicholoroacetic acid, 2-Deoxy-D-glucose and Metformin on cell cycle and recovery of Osteosarcoma cells were assessed. Their sensitivity to chemotherapy was also assessed when treated in combination with electroporation-delivered metabolic modulators.

ICOS activation in combination with electrochemotherapy generates effective anti-cancer immunological responses in murine models of primary, secondary and metastatic disease.

Electrochemotherapy is an evolving therapy which has recently been shown to induce an immunogenic form of cell death. It is hypothesized that the immunogenic cell death induced by electrochemotherapy may compliment the responses seen with anti-cancer immunotherapies. We therefore examined the effect of electrochemotherapy in combination with ICOS activation, which promotes the activity of previously activated T cells. In comparison to either monotherapy which resulted in no curative outcomes in any model, in a CT26 primary tumour 50% of mice were cured, with 100% of cured mice surviving tumour rechallenge. In a dual flank CT26 model mimicking secondary disease 20% of mice were cured, and 30% of mice were cured using an aggressively metastatic Lewis Lung Carcinoma model. We have shown the novel combination of electrochemotherapy with ICOS activation can inhibit local and distal tumour growth, including total tumour clearance with long lasting immunological memory.

Expressional changes in stemness markers post electrochemotherapy in pancreatic cancer cells.

Pancreatic cancer is one of the most lethal cancers with high metastatic potential and strong chemoresistance. The capability of a tumor to grow and propagate is dependent on a small subset of cells within a tumor, termed cancer stem cells. Cancer stem cells exhibit great tumorigenicity and are closely correlated with drug resistance and tumor recurrence. The aim of our study was to illustrate electrochemotherapy as an effective treatment for pancreatic cancer along with the expression change in stemness genes (Nanog, Sox2 and Oct3/4) in pancreatic cancer cells post electrochemotherapy with bleomycin, cisplatin and oxaliplatin. Our results showed the enhanced expression of Nanog and decreased expression level of Oct3/4 after electrochemotherpy. We thus propose that these stemness markerS may have important roles in the initiation and/or recurrence of pancreatic cancer, and consequently may serve as important molecular diagnostics and/or therapeutic targets for the development of novel treatment strategies in pancreatic cancer patients. In conclusion, targeting these stemness factors could potentially improve electrochemotherapy as a treatment and preventing recurrence.

Calcium electroporation induces tumor eradication, long-lasting immunity and cytokine responses in the CT26 colon cancer mouse model.

Electroporation is used in cancer treatment because of its ability to increase local cytotoxicity of e.g. bleomycin (electrochemotherapy) and calcium (calcium electroporation). Calcium electroporation is a novel anticancer treatment that selectively kills cancer cells by necrosis, a cell death pathway that stimulates the immune system due to high release of antigens and "danger signals." In this exploratory study, we aimed to investigate whether calcium electroporation could initiate an anticancer immune response similar to electrochemotherapy. To this end, we treated immunocompetent balb/c mice with CT26 colon tumors with calcium electroporation, electrochemotherapy, or ultrasound-based delivery of calcium or bleomycin. High treatment efficiency was observed with 100% complete remission in all four groups (12/12 with complete remission in each treatment group). In addition, none of the surviving mice from these groups formed new tumors when re-challenged with CT26 cancer cells 100-d post treatment, whereas mice challenged with different cancer cells (4T1 breast cancer) all developed tumors. Treatment of immunodeficient mice with calcium electroporation and electrochemotherapy showed no long-lasting tumor response. Calcium electroporation and electrochemotherapy was associated with a release of High Mobility Group Box 1 protein (HMGB1) in vitro (p = 0.029) and a significant increase of the overall systemic level of pro-inflammatory cytokines in serum from the treated mice (p < 0.003). These findings indicate that calcium electroporation as well as electrochemotherapy could have a role as immune stimulators in future treatments.