A peer-reviewed journal would not survive without the generous time and insightful comments of the reviewers, whose efforts often go unrecognized. Although final decisions are always editorial, they are greatly facilitated by the deeper technical knowledge, scientific insights, understanding of social consequences, and passion that reviewers bring to our deliberations. For these reasons, the Editor-in-Chief and staff of the journal warmly thank the reviewers whose comments helped to shape Biological Procedures Online, for their invaluable assistance with review of manuscripts for the journal in Volume 16 (2014).
Delivering genes and other materials directly into the tumor tissue causes specifically localized and powerfully enhanced efficacy of treatments; however, these specific effects can cause rapid, drastic changes in the appearance, texture, and consistency of the tumor. These changes complicate clinical response measurements which can confound the results and render recurring treatments difficult to perform and clinical response measurements nearly impossible to accurately obtain. One of these complicating issues is local swelling. Here, we will demonstrate how swelling caused by intratumoral gene treatments can confound the clinical results and impede further treatments, and we will demonstrate an easy technique to help to overcome this potential hurdle.
canine; oral tumor; intratumoral; electroporation; gene therapy; chemotherapy; chemogene therapy; immune therapy; IL12
Electrical pulses directly and effectively boost both in vitro and in vivo gene transfer, but this process is greatly affected by non-electrical factors that exist during electroporation. These factors include, but are not limited to, the types of cells or tissues used, the property of DNA, DNA formulation, and the expressed protein. In this mini-review, we only describe and discuss a summary of DNA properties and selected DNA formulations on gene transfer via electroporation. The properties of DNA were selected for review because a substantial amount of remarkable work has been performed during the past few years but has received less notice than other work, although DNA properties appear to be critical for boosting electroporation delivery. The selected formulations will be covered in this mini-review because we are only interested in the simple formulations that could be used for cell or gene therapy via electroporation. Plus, there was an extensive review of DNA formulations in the first edition of this book. The formulations discussed in this mini-review represent novel developments in recent years and may impact electroporation significantly. These advancements in DNA formulations could prove to be important for gene delivery and disease treatment.
Cell transfection efficiency often determines the success of cell-based gene therapy. Cell transfection via Nucleofector technology yields high transfection efficiency and low cytotoxicity. However, owing to trade secrecy, the components in each buffer are unknown, which not only increases the cost of electroporation studies but also limits the application of Nucelofector in clinical cell-based gene therapies. Thus, we developed a three-step method to determine the optimal conditions, including buffer, program and additional polymer, in electroporation for multiple cancers and stem cell lines. This method could reduce the cost, allow researchers to find the optimal electroporation conditions for their cell lines of interest, and greatly boost the application potential of electroporation in clinical cell-based gene therapies.
The ability to control the immune system to actively attack tumors would be a marvelous weapon to combat the incessant attack of cancer. Unfortunately, safe and effective methods are not yet available. To overcome the impediments to this control, tumor-targeted (tt) Interleukin (IL) 12 plasmid DNA can be safely delivered to accessible tumors, and these treatments can induce antitumor immune responses in both the treated and untreated tumors. Here, electroporation-mediated ttIL12 pDNA treatments are shown to be safe and well tolerated in a dose escalation study in canines bearing naturally-occurring neoplasms. The final patient received treatment with up to 3,800 μg pDNA distributed throughout five separate squamous cell carcinoma tumors, doses equivalent to those administered in a Phase I trial with wildtype IL12 pDNA. Not a single severe adverse event occurred in any patient at any of the five dose levels, and only minor, transient changes were noted in any tested parameter. Clinical response analysis and immune marker mRNA detection of treated and non-treated lesions confirmed that the ttIL12 pDNA treatments in only a few tumors could elicit antitumor immune responses in the treated lesions as well as distant metastatic lesions. These observations and results prove that ttIL12 pDNA can be safely administered at clinical levels, and these treatments can effect both treated and non-treated, metastatic lesions.
Cancer; Canine; Electroporation; Gene therapy; Immunotherapy; Interleukin 12; Tumor-targeting
For decades, immortal cancer cell lines have constituted an accessible, easily usable set of biological models to investigate cancer biology and explore the potential efficacy of anticancer drugs. However, numerous studies suggest that these cell lines poorly represent the diversity, heterogeneity and drug-resistant tumors occurring in patients. The derivation and short -term culture of primary cells from solid tumors have thus gained significant importance in personalized cancer therapy. This review focuses our current understanding and the pros and cons of different methods for primary tumor cell culture. Furthermore, various culture matrices like biomimetic scaffolds, chemically defined media supplemented with essential nutrients have been prepared for different tissues. These well characterized primary tumor cells redefine cancer therapies with high translational relevance.
Personalized therapy; primary tumor cell lines
Increased infiltration of CD8+T cells into tumors has a positive impact on survival. Our previous study showed that doxorubicin (Dox) plus interleukin-12 (IL-12) boosted the accumulation of CD8+T cells in tumors and had a greater antitumor effect than did either agent alone. The purpose of this study was to determine the impact of NKG2D expression on CD8+T cell infiltration and antitumor efficacy.
Tumor-bearing mice were administered Dox, IL-12 plasmid DNA, or both via intraperitoneal injection or intramuscular electroporation. The induction of NKG2D on CD8+T cells and other lymphocytes was analyzed via flow cytometry, and NKG2D-positive CD8+T cell–specific localization in tumors was determined by using immunofluorescence staining in various types of immune cell–depleted mice.
The combination of Dox plus IL-12 specifically increased expression of NKG2D in CD8+T cells but not in other types of immune cells, including NK cells, which naturally express NKG2D. This induced NKG2D expression in CD8+T cells was associated with increased accumulation of CD8+T cells in murine tumors. Administration of NKG2D-blocking antibody or CD8+T cell–depletion antibody abrogated the NKG2D+CD8+T cell detection in tumors, whereas administration of NK cell–depletion antibody had no effect. Increased NKG2D expression in CD8+T cells was associated with increased antitumor efficacy in vivo.
We conclude that Dox plus IL-12 induces NKG2D in CD8+T cells in vivo and boosts NKG2D+CD8+T-dependent antitumor immune surveillance. This discovery reveals a novel mechanism for how chemoimmunotherapy synergistically promotes T cell–mediated antitumor immune surveillance.
Interleukin-12; Doxorubicin; Tumor-infiltrating lymphocytes; NKG2D+CD8+T cells
Although genetically engineered cells have been used to generate monoclonal antibodies (mAbs) against numerous proteins, no study has used them to generate mAbs against glycosylphosphatidylinositol (GPI)-anchored proteins. The GPI-linked protein Rae-1, an NKG2D ligand member, is responsible for interacting with immune surveillance cells. However, very few high-quality mAbs against Rae-1 are available for use in multiple analyses, including Western blotting, immunohistochemistry, and flow cytometry. The lack of high-quality mAbs limits the in-depth analysis of Rae-1 fate, such as shedding and internalization, in murine models. Moreover, currently available screening approaches for identifying high-quality mAbs are excessively time-consuming and costly.
We used Rae-1–overexpressing CT26 tumor cells to generate 60 hybridomas that secreted mAbs against Rae-1. We also developed a streamlined screening strategy for selecting the best anti–Rae-1 mAb for use in flow cytometry assay, enzyme-linked immunosorbent assay, Western blotting, and immunostaining.
Our cell line–based immunization approach can yield mAbs against GPI-anchored proteins, and our streamlined screening strategy can be used to select the ideal hybridoma for producing such mAbs.
GPI-anchored protein Rae-1; Monoclonal antibody; Hybridomas; Streamlined screening strategy
Bone marrow-derived cells have a potent impact on the formation and progression of tumor metastasis. This study demonstrates that bone marrow directly promotes metastasis to distant sites from tumor cells residing in the bone marrow in multiple types of tumors and multiple tumor cell lines. The bone marrow environment requires less tumor cells for inducing distant metastasis and overcomes the inhibition of metastasis resulting from engineering the tumor cells with reporter genes. This discovery provides an effective approach to generate spontaneous-like metastatic tumor models which will satisfy the urgent need for studying metastasis biology and discovering novel therapeutics.
Metastasis; Bone Marrow; Mouse models; Metastatic Cascade
IL-27 is a member of the IL-12 family of cytokines that is comprised of an IL-12 p40-related protein subunit, EBV-induced gene 3 (EBI3), and a p35-related subunit, p28. IL-27 functions through IL-27R and has been shown to have potent anti-tumor activity via activation of a variety of cellular components, including anti-tumor CD8+ T-cell responses. However, the exact mechanisms of how IL-27 enhances anti-tumor CD8+ T-cell responses remain unclear. Here we show that IL-27 significantly enhances the survival of activated tumor antigen specific CD8+ T cells in vitro and in vivo, and programs tumor antigen-specific CD8+ T cells into memory precursor (MPC)-like effector cells, characterized by upregulation of Bcl-6, SOCS3, Sca-1, and IL-10. While STAT3 activation and the CTL survival-enhancing effects can be independent of CTL IL-10 production, we show here that IL-27-induced CTL IL-10 production contributes to MPC phenotype induction, CTL memory, and tumor rejection. Thus, IL-27 enhances anti-tumor CTL responses via programming tumor antigen-specific CD8+ T cells into a unique memory precursor-type of effector cells characterized by a greater survival advantage. Our results have important implications for designing immunotherapy against human cancer.
Studies into the regulation of interleukin-10 (IL-10), have focused only on the molecular or single-cell level. The cues that induce IL-10 in the context of cell-to-cell communication are scarce. To fill this gap, this study elucidates the cell-to-cell interaction dependent regulation of IL-10.
The simultaneous activation of CD4+ T cells via CD3/CD28 and stimulation of macrophages via CpG and their intercellular communication with each other in the same microenvironment is necessary to induce a synergistic expression of IL-10. NF-κB1, ERK, and STAT3 are positive regulators of this cell-to-cell communication mediated molecular change of IL-10 induction. Strikingly, the activation of CD40/CD154 signaling is a negative regulator of IL-10 levels by CD3/CD28/CpG.
These findings are of prominence as CD3/CD28/CpG treatment can induce the anti-inflammatory cytokines IL-10 and IL-30, and the activation or inhibition of the CD40/CD154 acts as molecular rheostat of the expression of IL-10 or IL-30. More importantly, this not only serves as an example of IL-10 regulation at the cellular via coordination of two signals from two cell types, but these findings also lay the molecular and cellular groundwork for future studies to investigate how to manipulate IL-10 or IL-30 production during inflammation, cancer, or autoimmune diseases.
IL-10; IL27p28 (IL-30); NF-κB1; STAT3; CD40/CD154; CD4+ T cells; pERK; F4/80 cells
The safest and most effective cytokine therapies require the favorable accumulation of the cytokine in the tumor environment. While direct treatment into the neoplasm is ideal, systemic tumor-targeted therapies will be more feasible. Electroporation-mediated transfection of cytokine plasmid DNA including a tumor-targeting peptide-encoding sequence is one method for obtaining a tumor-targeted cytokine produced by the tumor-bearing patient's tissues. Here, the impact on efficacy of the location of targeting peptide, choice of targeting peptide, tumor histotype, and cytokine utilization are studied in multiple syngeneic murine tumor models. Within the same tumor model, the location of the targeting peptide could either improve or reduce the antitumor effect of interleukin (IL)12 gene treatments, yet in other tumor models the tumor-targeted IL12 plasmid DNAs were equally effective regardless of the peptide location. Similarly, the same targeting peptide that enhances IL12 therapies in one model fails to improve the effect of either IL15 or PF4 for inhibiting tumor growth in the same model. These interesting and sometimes contrasting results highlight both the efficacy and personalization of tumor-targeted cytokine gene therapies while exposing important aspects of these same therapies which must be considered before progressing into approved treatment options.
RBC membrane-cloaked polymeric nanoparticles represent an emerging nanocarrier platform with extended circulation in vivo. A lipid-insertion method is employed to functionalize these nanoparticles without the need for direct chemical conjugation. Insertion of both folate and the nucleolin-targeting aptamer AS1411 shows receptor-specific targeting against model cancer cell lines.
Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers worldwide. It arises from modulation of multiple genes by mutations, epigenetic regulation, noncoding RNAs and translational modifications of encoded proteins. Although >40% of HCCs are clonal and thought to arise from cancer stem cells (CSCs), the precise identification and mechanisms of CSC formation remain poorly understood. A functional role of transforming growth factor (TGF)-β signalling in liver and intestinal stem cell niches has been demonstrated through mouse genetics. These studies demonstrate that loss of TGF-β signalling yields a phenotype similar to a human CSC disorder, Beckwith–Wiedemann syndrome. Insights into this powerful pathway will be vital for developing new therapeutics in cancer. Current clinical approaches are aimed at establishing novel cancer drugs that target activated pathways when the TGF-β tumour suppressor pathway is lost, and TGF-β itself could potentially be targeted in metastases. Studies delineating key functional pathways in HCC and CSC formation could be important in preventing this disease and could lead to simple treatment strategies; for example, use of vitamin D might be effective when the TGF-β pathway is lost or when wnt signalling is activated.
The presence of the Philadelphia chromosome in patients with acute lymphoblastic leukemia (Ph+ALL) is a negative prognostic indicator. Tyrosine kinase inhibitors (TKI) that target BCR/ABL, such as imatinib, have improved treatment of Ph+ALL and are generally incorporated into induction regimens. This approach has improved clinical responses, but molecular remissions are seen in less than 50% of patients leaving few treatment options in the event of relapse. Thus, identification of additional targets for therapeutic intervention has potential to improve outcomes for Ph+ALL. The human epidermal growth factor receptor 2 (ErbB2) is expressed in ∼30% of B-ALLs, and numerous small molecule inhibitors are available to prevent its activation. We analyzed a cohort of 129 ALL patient samples using reverse phase protein array (RPPA) with ErbB2 and phospho-ErbB2 antibodies and found that activity of ErbB2 was elevated in 56% of Ph+ALL as compared to just 4.8% of Ph−ALL. In two human Ph+ALL cell lines, inhibition of ErbB kinase activity with canertinib resulted in a dose-dependent decrease in the phosphorylation of an ErbB kinase signaling target p70S6-kinase T389 (by 60% in Z119 and 39% in Z181 cells at 3 µM). Downstream, phosphorylation of S6-kinase was also diminished in both cell lines in a dose-dependent manner (by 91% in both cell lines at 3 µM). Canertinib treatment increased expression of the pro-apoptotic protein Bim by as much as 144% in Z119 cells and 49% in Z181 cells, and further produced caspase-3 activation and consequent apoptotic cell death. Both canertinib and the FDA-approved ErbB1/2-directed TKI lapatinib abrogated proliferation and increased sensitivity to BCR/ABL-directed TKIs at clinically relevant doses. Our results suggest that ErbB signaling is an additional molecular target in Ph+ALL and encourage the development of clinical strategies combining ErbB and BCR/ABL kinase inhibitors for this subset of ALL patients.
Interleukin 30 (IL30), the p28 subunit of IL27, interacts with Epstein-Barr virus induced gene 3 to form IL27, which modulates both pro- and anti-inflammatory responses during autoimmune or infectious disease. It also acts as a natural antagonist of glycoprotein 130 (gp130), thereby attenuating the signals of other gp130-associated cytokines. IL30 regulation via LPS has been reported by others, but the intercellular communication that induces IL30 expression is unknown. Here, we show that treatment with anti-CD3/CD28 antibodies plus CpG oligodeoxynucleotides induces robust expression of IL30, whereas either treatment alone induces only low expression of IL30. This observation in vitro mirrors murine model in which administration of CpG under inflammatory conditions in vivo induces IL30 expression. This robust induction of IL30 occurs through the coordination of helper CD4+ T cells and innate immune cells (such as macrophages) and, to a lesser degree, B cells via the CD40/CD154 signaling pathway. These findings reveal a previously unrecognized mechanism that integrates signaling pathways from T cells and macrophages at the cellular level to induce IL30 expression.
The liver is the major metabolic organ and is subjected to constant attacks from chronic viral infection, uptake of therapeutic drugs, life behavior (alcoholic), and environmental contaminants, all of which result in chronic inflammation, fibrosis, and ultimately, cancer. Therefore, there is an urgent need to discover effective therapeutic agents for the prevention and treatment of liver injury; the ideal drug being a naturally occurring biological inhibitor. Here, we establish the role of IL30 as a potent anti-inflammatory cytokine which can inhibit inflammation-induced liver injury. In contrast, IL27, which contains IL30 as a subunit, is not hepatoprotective. Interestingly, IL30 is induced by the pro-inflammatory signal such as IL12 through IFNγ/STAT1 signaling. In animal models, administration of IL30 via a gene therapy approach prevents and treats both IL12-, IFNγ-, and Concanavalin A -induced liver toxicity. Likewise, immunohistochemistry analysis of human tissue samples revealed that IL30 is highly expressed in hepatocytes yet barely expressed in inflammation-induced tissue such as fibrous/connective tissue. These novel observations reveal a novel role of IL30 as a therapeutic cytokine that suppresses pro-inflammatory cytokine-associated liver toxicity.
IL12; IFNγ; IL27p28 (IL30); EBI3; TCCR; ConA
Vimentin, a major constituent of the intermediate filament (IF) family of proteins, is ubiquitously expressed in normal mesenchymal cells and is known to maintain cellular integrity and provide resistance against stress. Increased vimentin expression has been reported in various epithelial cancers including prostate cancer, gastrointestinal tumors, CNS tumors, breast cancer, malignant melanoma, lung cancer and other types of cancers. Vimentin's over-expression in cancer correlates well with increased tumor growth, invasion and poor prognosis; however, the role of vimentin in cancer progression remains obscure. In the recent years, vimentin has gained much importance as a marker for epithelial-mesenchymal transition (EMT). Although EMT is associated with a number of tumorigenic events, the role of vimentin in the underlying events mediating these processes remains unknown. Though majority of the literature findings indicate a future significance of vimentin as a biomarker for different cancers with clinical relevance, more research in to the molecular aspects will be crucial to particularly evaluate the function of vimentin in the process of tumorigenesis. By virtue of its over-expression in a large number of cancers and its role in mediating various tumorigenic events, vimentin serves as an attractive target for cancer therapy. Further, research directed toward elucidating the role of vimentin in various signaling pathways would open up new approaches for the development of promising therapeutic agents. This review summarizes the expression and functions of vimentin in cancers and also suggests some directions toward future cancer therapy utilizing vimentin as a potential target.
This study’s goal was to assess the safety of tumor-targeted interleukin-12 (ttIL-12) when administered by electrogenetherapy in C3H/HeJ mice by identifying an initial safe dose for human dose escalation schemes, toxicity target organs, markers of toxicity, and toxicity reversibility. Tumor-free mice receiving two doses of 0.45% NaCl, 1 µg ttIL-12 DNA in 0.45% NaCl, or 5 µg ttIL-12 DNA in 0.45% NaCl ten days apart combined with low-intensity electroporation were compared to non-treatment controls over time. All mice had blood cell counts, serum chemistry profiles, plasma IL-12 and IFNγ determinations, necropsy, and multi-organ histopathology. Mild treatment-associated changes included electroporation-associated muscle changes that resolved by 30 days; decreased total white blood cell counts and infectious disease in the 5 µg ttIL-12 group, but not in the 1 µg group; and liver changes in ttIL-12 groups that correlated with alanine transaminase (ALT) levels and resolved by 30 days. Dystrophic cardiac calcification seen in older, 5 µg ttIL-12-treated mice was the only serious toxicity. Based on these results and the lack of any effect on wound healing when combined with surgery, low-intensity electrogenetherapy with ttIL-12 appears to be safe and well tolerated.
Gene therapy; IL-12; tumor targeting; toxicity; cancer; electroporation
Established cell transfection via nucleofection relies on nucleofection buffers with unknown and proprietary makeup due to trade secrecy, inhibiting the possibility of using this otherwise effective method for developing cell therapy. We devised a three-step method for discovering an optimal formulation for the nucleofection of any cell-line. These steps include the selection of the best nucleofection program and known buffer type, selection of the best polymer for boosting the transfection efficiency of the best buffer, and the comparison with the optimal buffer from an established commercial vendor (Amaxa). Using this 3-step selection system, competitive nucleofection formulations were discovered for multiple cell lines, which are equal to or surpass the efficiency of the Amaxa nucleofector solution in a variety of cells and cell lines, including primary adipose stem cells, muscle cells, tumor cells, and immune cells. Through the use of scanning electron microscopy, we have revealed morphological changes, which predispose for the ability of these buffers to assist in transferring plasmid DNA into the nuclear space. Our formulation may greatly reduce the cost of electroporation study in laboratory and boosts the potential of application of electroporation-based cell therapies in clinical trials.
electroporation; cell transfection; cell therapy; adipose stem cells; formulation
Adipose stem cells have a strong potential for use in cell-based therapy, but the current nucleofection technique, which relies on unknown buffers, prevents their use.
We developed an optimal nucleofection formulation for human adipose stem cells by using a three-step method that we had developed previously. This method was designed to determine the optimal formulation for nucleofection that was capable of meeting or surpassing the established commercial buffer (Amaxa), in particular for murine adipose stem cells. By using this same buffer, we determined that the same formulation yields optimal transfection efficiency in human mesenchymal stem cells.
Our findings suggest that transfection efficiency in human stem cells can be boosted with proper formulation.
Electroporation; Formulation; Stem cells; Transfection; Cell therapy
Tumor-targeted antibody therapy is one of the safest biological therapeutics for cancer patients, but it is often ineffective at inducing direct tumor cell death and is ineffective against resistant tumor cells. Currently, the antitumor efficacy of antibody therapy is primarily achieved by inducing indirect tumor cell death, such as antibody-dependent cell cytotoxicity. Our study reveals that Herceptin conjugates, if generated via the crosslinker EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), are capable of engendering human epidermal growth factor receptor 2 (Her2) positive tumor cells death. Using a high-performance liquid chromatography (HPLC) system, three peaks with estimated molecular weights of antibody monomer, dimer, and trimer were isolated. Both Herceptin trimer and dimer separated by HPLC induced significant levels of necrotic tumor cell death, although the trimer was more effective than the dimer. Notably, the Herceptin trimer also induced Herceptin-resistant tumor cell death. Surprisingly different from the known cell death mechanism that often results from antibody treatment, the Herceptin trimer elicited effective and direct tumor cell death via a novel mechanism: programmed cell necrosis. In Her2-positive cells, inhibition of necrosis pathways significantly reversed Herceptin trimer-induced cell death. In summary, the Herceptin trimer reported herein harbors great potential for overcoming tumor cell resistance to Herceptin treatment.
Based on superior outcomes from electrochemogene therapy (ECGT) compared to electrochemotherapy (ECT)in mice, we determined the efficacy of ECGT applied to spontaneous canine neoplasms. Intralesional bleomycin (BLM) and feline interleukin-12 DNA (fIL-12 DNA) injection combined with trans-lesional electroporation resulted in complete cure of two recurrent World Health Organization (WHO) stageT2bN0M0 oral squamous cell carcinomas (SCCs)and one T2N0M0acanthomatous ameloblastoma. Three remaining dogs, which had no other treatment options, had partial responses to ECGT; one had mandibular T3bN2bM1 melanoma with pulmonary and lymph node metastases; one had cubital T3N0M1 histiocytic sarcoma with spleen metastases; and one had soft palate T3N0M0fibrosarcoma. The melanoma dog had decrease in the size of the primary tumor before recrudescence and euthanasia. The histiocytic sarcoma dog had resolution of the primary tumor, but was euthanized because of metastases four months after the only treatment. The dog with T 3N0M0 fibrosarcoma had tumor regression with recrudescence. Treatment was associated with minimal side effects and was easy to perform; was associated with repair of bone lysis in cured dogs; improved quality of life for dogs with partial responses; and extended overall survival time. ECGT appears to be a safe and resulted in complete responses in SCC and acanthomatous ameloblastoma.
Electroporation; Bleomycin; IL-12; Canine; Neoplasia; Gene therapy
Crosstalk between tumor cells and the cognate microenvironment plays a crucial role in tumor initiation and progression. However, only a few genes are known to affect such a crosstalk. This study reveals that WSX1 plays such a role when highly expressed in tumor cells. The expression of WSX1 in Lewis Lung Carcinoma (LLC) and the melanoma cell line AGS induces the death of T cells and inhibits the production of the effector cytokine IFNγ from NK and T cells, resulting in the promotion of tumor growth. These pro-tumorigenic properties of WSX1 are independent of IL27. This key observation reveals a new pathway of tumor-host interaction, which will ultimately lead to better strategies in immune therapy to reverse tumor tolerance.
Eradication of residual malignancies and metastatic tumors via a systemic approach is the key for successfully treating cancer and increasing the cancer patient survival. Systemic administration of IL12 protein in an acute large dose is effective but toxic. Systemic administration of IL12 gene by persistently expressing a low level of IL12 protein may reduce the systemic toxicity, but only eradicates IL12 sensitive tumors. Here, we discovered that sequential administration of IL12 and IL27 encoding DNA, referred to as sequential IL12-IL27 gene therapy, not only eradicated IL12 sensitive tumors from 100% of mice but also eradicated the highly malignant 4T1 tumors from 33% of treated mice in multiple independent experiments. This IL12-IL27 sequential gene therapy is not only superior to IL12-IL12 sequential gene therapy for eliminating tumors, but also for inducing CTL activity, increasing T cell infiltration into tumors, and yielding a large number of tumor-specific IFNγ positive CD8 T cells. Notably, depletion of either T- or NK-cells during the IL27 treatment phase reverses tumor eradication, suggesting an NK-cell requirement for this sequential gene therapy-mediated tumor eradication. Both reversal of the administration sequence and co-administration of IL12 and IL27 impaired the tumor eradication in 4T1 tumor bearing mice. This IL12-IL27 sequential gene therapy, via sequential administration of IL12 and IL27 encoding plasmid DNA into tumor-bearing mice through intramuscular electroporation, provides a simple but effective approach for eliminating inaccessible residual tumors.
IL12; IL27; electroporation; gene therapy; tumors; T cells