Jeffry Cutrera

Universal Marker and Detection Tool for Human Sarcoma Circulating Tumor Cells

To date, no specific marker exists for the detection of circulating tumor cells (CTC) from different types of sarcomas, though tools are available for detection of CTCs in peripheral blood of patients with cancer for epithelial cancers. Here, we report cell-surface vimentin (CSV) as an exclusive marker on sarcoma CTC regardless of the tissue origin of the sarcoma as detected by a novel monoclonal antibody. Utilizing CSV as a probe, we isolated and enumerated sarcoma CTCs with high sensitivity and specificity from the blood of patients bearing different types of sarcoma, validating their phenotype by single cell genomic amplification, mutation detection, and FISH. Our results establish the first universal and specific CTC marker described for enumerating CTCs from different types of sarcoma, thereby providing a key prognosis tool to monitor cancer metastasis and relapse.

Interleukin-30: A novel antiinflammatory cytokine candidate for prevention and treatment of inflammatory cytokine-induced liver injury

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 antiinflammatory cytokine that can inhibit inflammation‐induced liver injury. In contrast, interleukin (IL)27, which contains IL30 as a subunit, is not hepatoprotective. Interestingly, IL30 is induced by the proinflammatory signal such as IL12 through interferon‐gamma (IFN‐γ) / signal transducer and activator of transcription 1 signaling. In animal models, administration of IL30 by way of a gene therapy approach prevents and treats both IL12‐, IFN‐γ‐, 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. Conclusion: These novel observations reveal a novel role of IL30 as a therapeutic cytokine that suppresses proinflammatory cytokine‐associated liver toxicity. (Hepatology 2012)

Bleomycin/interleukin-12 electrochemogene therapy for treating naturally occurring spontaneous neoplasms in dogs

On the basis of superior outcomes from electrochemogene therapy (ECGT) compared with electrochemotherapy in mice, we determined the efficacy of ECGT applied to spontaneous canine neoplasms. Intralesional bleomycin and feline interleukin-12 DNA (fIL-12 DNA) injection combined with translesional electroporation resulted in complete cure of two recurrent World Health Organization stage T2bN0M0 oral squamous cell carcinomas (SCCs) and one T2N0M0 acanthomatous 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 T3N0M0 fibrosarcoma. The melanoma dog had decrease in 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 4 months after the only treatment. The dog with T3N0M0 fibrosarcoma had tumor regression with recrudescence. Treatment was associated with minimal side effects and was easy to perform. It was associated with repair of bone lysis in cured dogs, it improved quality of life of dogs with partial responses and extended overall survival time. ECGT seems to be a safe and resulted in complete responses in SCC and acanthomatous ameloblastoma.

Discovery of a Linear Peptide for Improving Tumor Targeting of Gene Products and Treatment of Distal Tumors by IL-12 Gene Therapy

Like many effective therapeutics, interleukin-12 (IL-12) therapy often causes side effects. Tumor targeted delivery may improve the efficacy and decrease the toxicity of systemic IL-12 treatments. In this study, a novel targeting approach was investigated. A secreted alkaline phosphatase (SEAP) reporter gene-based screening process was used to identify a mini-peptide which can be produced in vivo to target gene products to tumors. The coding region for the best peptide was inserted into an IL-12 gene to determine the antitumor efficacy. Affinity chromatography, mass spectrometry analysis, and binding studies were used to identify a receptor for this peptide. We discovered that the linear peptide VNTANST increased the tumor accumulation of the reporter gene products in five independent tumor models including one human xenogeneic model. The product from VNTANST-IL-12 fusion gene therapy increased accumulation of IL-12 in the tumor environment, and in three tumor models, VNTANST-IL-12 gene therapy inhibited distal tumor growth. In a spontaneous lung metastasis model, inhibition of metastatic tumor growth was improved compared to wild-type IL-12 gene therapy, and in a squamous cell carcinoma model, toxic liver lesions were reduced. The receptor for VNTANST was identified as vimentin. These results show the promise of using VNTANST to improve IL-12 treatments.

Intratumoral bleomycin and IL-12 electrochemogenetherapy for treating head and neck tumors in dogs.

Bleomycin and Interleukin 12 have been used clinically to treat tumors; however, the co-administration of Bleomycin and Interleukin 12 followed by electroporation has not been tested clinically. In this study, dogs with spontaneous head and neck tumors were treated with one co-administration of Bleomycin and Interleukin 12 plasmid DNA followed by electroporation. The regression of the recurrent papillary tumor and the adjacent metastatic bone tumor was analyzed by multiple CT scans. The papillary tumor was completely eradicated in less than 2 weeks, and the bone tumor was not visible 23 weeks after the administration.

Expression of WSX1 in tumors sensitizes IL-27 signaling-independent natural killer cell surveillance.

It is well known that the interleukin (IL)-27 receptor WSX1 is expressed in immune cells and induces an IL-27-dependent immune response. Opposing this conventional dogma, this study reveals a much higher level of WSX1 expression in multiple types of epithelial tumor cells when compared with normal epithelial cells. Expression of exogenous WSX1 in epithelial tumor cells suppresses tumorigenicity in vitro and inhibits tumor growth in vivo. Different from the role of WSX1 in immune cells, the antitumor activity of WSX1 in epithelial tumor cells is independent of IL-27 signaling but is mainly dependent on natural killer (NK) cell surveillance. Deficiency of either the IL-27 subunit EBV-induced gene 3 or the IL-27 receptor WSX1 in the host animals had no effect on tumor growth inhibition induced by WSX1 expression in tumor cells. Expression of WSX1 in epithelial tumor cells enhances NK cell cytolytic activity against tumor cells, whereas the absence of functional NK cells impairs the WSX1-mediated inhibition of epithelial tumor growth. The underlying mechanism by which WSX1 expression in tumor cells enhances NK cytolytic activity is dependent on up-regulation of NKG2D ligand expression. Our results reveal an IL-27-independent function of WSX1: sensitizing NK cell-mediated antitumor surveillance via a NKG2D-dependent mechanism.

Administering Plasmid DNA Encoding Tumor Vessel–anchored IFN-α for Localizing Gene Product Within or Into Tumors

Tumor-targeted gene delivery has been intensively studied in the field of gene therapy, but no attention has been given to targeting the therapeutic gene products, which are transcribed and translated from the injected genes, into tumors. Targeting immune stimulatory gene products into tumors is the key to triggering tumor-specific CD8(+) T-cell responses and reducing systemic toxicity. To target the gene products generated from the injected genes into tumors, genes encoding the tumor-targeted fusion gene product were generated and administered locally and systemically via electroporation. As anticipated, administration of a therapeutic gene encoding IFN-alpha and the tumor vessel-targeted peptide CDGRC fusion gene product minimizes the leakage of immunostimulatory cytokine from tumors into the blood circulation, increases the infiltration of CD8(+) T cells into tumors, induces a high magnitude of cytotoxic T-cell lysis (CTL) activity, and reduces tumor vessel density. As a result, tumor growth was more significantly inhibited by administering the IFN-alpha-CDGRC gene than by administering the wild-type IFN-alpha gene. The same result was obtained with the systemic administration of the tumor-targeted IFN-alpha gene. This gene product-based tumor-targeted gene therapy approach could complement any other tumor-targeted gene delivery method for improving tumor-targeting efficiency.Tumor-targeted gene delivery has been intensively studied in the field of gene therapy, but no attention has been given to targeting the therapeutic gene products, which are transcribed and translated from the injected genes, into tumors. Targeting immune stimulatory gene products into tumors is the key to triggering tumor-specific CD8+ T-cell responses and reducing systemic toxicity. To target the gene products generated from the injected genes into tumors, genes encoding the tumor-targeted fusion gene product were generated and administered locally and systemically via electroporation. As anticipated, administration of a therapeutic gene encoding IFN-α and the tumor vessel-targeted peptide CDGRC fusion gene product minimizes the leakage of immunostimulatory cytokine from tumors into the blood circulation, increases the infiltration of CD8+ T cells into tumors, induces a high magnitude of cytotoxic T-cell lysis (CTL) activity, and reduces tumor vessel density. As a result, tumor growth was more significantly inhibited by administering the IFN-α-CDGRC gene than by administering the wild-type IFN-α gene. The same result was obtained with the systemic administration of the tumor-targeted IFN-α gene. This gene product-based tumor-targeted gene therapy approach could complement any other tumor-targeted gene delivery method for improving tumor-targeting efficiency.

Coordination between TLR9 Signaling in Macrophages and CD3 Signaling in T Cells Induces Robust Expression of IL-30

IL-30, the p28 subunit of IL-27, interacts with EBV-induced gene 3 to form IL-27, which modulates both proinflammatory and anti-inflammatory responses during autoimmune or infectious disease. It also acts as a natural antagonist of gp130, thereby attenuating the signals of other gp130-associated cytokines. IL-30 regulation via LPS has been reported by others, but the intercellular communication that induces IL-30 expression is unknown. In this study, we show that treatment with anti-CD3/CD28 Abs plus CpG oligodeoxynucleotides induces robust expression of IL-30, whereas either treatment alone induces only low expression of IL-30. This observation in vitro mirrors the murine model in which administration of CpG under inflammatory conditions in vivo induces IL-30 expression. This robust induction of IL-30 occurs through the coordination of helper CD4+ T cells and innate immune cells (e.g., 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 IL-30 expression.

Safe and effective treatment of spontaneous neoplasms with interleukin 12 electro‐chemo‐gene therapy

Electroporation improves the anti‐tumour efficacy of chemotherapeutic and gene therapies. Combining electroporation‐mediated chemotherapeutics with interleukin 12 (IL‐12) plasmid DNA produces a strong yet safe anti‐tumour effect for treating primary and refractory tumours. A previously published report demonstrated the efficacy of a single cycle of IL‐12 plasmid DNA and bleomycin in canines, and, similarly, this study further demonstrates the safety and efficacy of repeated cycles of chemotherapy plus IL‐12 gene therapy for long‐term management of aggressive tumours. Thirteen canine patients were enrolled in this study and received multiple cycles of electro‐chemo‐gene therapy (ECGT) with IL‐12 pDNA and either bleomycin or gemcitabine. ECGT treatments are very effective for inducing tumour regression via an antitumour immune response in all tested histotypes except for sarcomas, and these treatments can quickly eradicate or debulk large squamous cell carcinomas. The versatility of ECGT allows for response‐based modifications which can overcome treatment resistance for affecting refractory lesions. Importantly, not a single severe adverse event was noted even in animals receiving the highest doses of chemotherapeutics and IL12 pDNA over multiple treatment cycles. This report highlights the safety, efficacy and versatility of this treatment strategy. The data reveal the importance of inducing a strong anti‐tumour response for successfully affecting not only the treated tumours, but also non‐treated metastatic tumours. ECGT with IL12 pDNA plus chemotherapy is an effective strategy for treating multiple types of spontaneous cancers including large, refractory and multiple tumour burdens.

Mutant p53 in concert with an interleukin‐27 receptor alpha deficiency causes spontaneous liver inflammation, fibrosis, and steatosis in mice

The cellular and molecular etiology of unresolved chronic liver inflammation remains obscure. Whereas mutant p53 has gain‐of‐function properties in tumors, the role of this protein in liver inflammation is unknown. Herein, mutant p53R172H is mechanistically linked to spontaneous and sustained liver inflammation and steatosis when combined with the absence of interleukin‐27 (IL27) signaling (IL27RA), resembling the phenotype observed in nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) patients. Indeed, these mice develop, with age, hepatocyte necrosis, immune cell infiltration, fibrosis, and micro‐ and macrosteatosis; however, these phenotypes are absent in mutant p53R172H or IL27RA‐/‐ mice. Mechanistically, endothelin A receptor (ETAR)‐positive macrophages are highly accumulated in the inflamed liver, and chemical inhibition of ETAR signaling reverses the observed phenotype and negatively regulates mutant p53 levels in macrophages. Conclusion: The combination of mutant p53 and IL27RA‐/‐ causes spontaneous liver inflammation, steatosis, and fibrosis in vivo, whereas either gene alone in vivo has no effects on the liver. (Hepatology 2016;63:1000–1012)