Role of ADCC, CDC, and CDCC in Vaccine-Mediated Protection against Her2 Mammary Carcinogenesis.

Amplification or mutation of the Her2 oncoantigen in human mammary glands leads to the development of an aggressive breast carcinoma. Several features of this breast carcinoma are reproduced in mammary carcinomas that spontaneously arise in female transgenic mice bearing the activated rat Her2 oncogene under transcriptional control of the mouse mammary tumor virus promoter-BALB-neuT (neuT) mice. We previously demonstrated that carcinoma progression in neuT mice can be prevented by DNA vaccination with RHuT, a plasmid coding for a chimeric rat/human Her2 protein. RHuT vaccination exerts an antitumor effect, mostly mediated by the induction of a strong anti-rat Her2 antibody response. IgG induced by RHuT vaccine mainly acts by blocking Her2 signaling, thus impairing cell cycle progression and inducing apoptosis of cancer cells, but other indirect effector mechanisms could be involved in the antibody-mediated protection. The recruitment of cells with perforin-dependent cytotoxic activity, able to perform antibody-dependent cellular cytotoxicity, has already been investigated. Less is known about the role of the complement system in sustaining antitumor response through complement-dependent cytotoxicity and cellular cytotoxicity in vaccinated mice. This work highlights that the weight of such mechanisms in RHuT-induced cancer protection is different in transplantable versus autochthonous Her2+ tumor models. These results may shed new light on the effector mechanisms involved in antibody-dependent anti-cancer responses, which might be exploited to ameliorate the therapy of Her2+ breast cancer.

Liver-Specific siRNA-Mediated Stat3 or C3 Knockdown Improves the Outcome of Experimental Autoimmune Myocarditis.

Myocarditis can lead to autoimmune disease, dilated cardiomyopathy, and heart failure, which is modeled in the mouse by cardiac myosin immunization (experimental autoimmune myocarditis [EAM]). Signal transducer and activator of transcription 3 (STAT3) systemic inhibition exerts both preventive and therapeutic effects in EAM, and STAT3 constitutive activation elicits immune-mediated myocarditis dependent on complement C3 and correlating with activation of the STAT3-interleukin 6 (IL-6) axis in the liver. Thus, liver-specific STAT3 inhibition may represent a therapeutic option, allowing to bypass the heart toxicity, predicted by systemic STAT3 inhibition. We therefore decided to explore the effectiveness of silencing liver Stat3 and C3 in preventing EAM onset and/or the recovery of cardiac functions. We first show that complement C3 and C5 genetic depletion significantly prevents the onset of spontaneous myocarditis, supporting the complement cascade as a viable target. In order to interfere with complement production and STAT3 activity specifically in the liver, we took advantage of liver-specific Stat3 or C3 small interfering (si)RNA nanoparticles, demonstrating that both siRNAs can significantly prevent myocarditis onset and improve the recovery of heart functions in EAM. Our data demonstrate that liver-specific Stat3/C3 siRNAs may represent a therapeutic option for autoimmune myocarditis and suggest that complement levels and activation might be predictive of progression to dilated cardiomyopathy.

Poly-specific neoantigen-targeted cancer vaccines delay patient derived tumor growth.

BACKGROUND: Personalized cancer vaccines based on neoantigens have reached the clinical trial stage in melanoma. Different vaccination protocols showed efficacy in preclinical models without a clear indication of the quality and the number of neoantigens required for an effective cancer vaccine. METHODS: In an effort to develop potent and efficacious neoantigen-based vaccines, we have developed different neoantigen minigene (NAM) vaccine vectors to determine the rules for a successful neoantigen cancer vaccine (NCV) delivered by plasmid DNA and electroporation. Immune responses were analyzed at the level of single neoantigen by flow cytometry and correlated with tumor growth. Adoptive T cell transfer, from HLA-2.1.1 mice, was used to demonstrate the efficacy of the NCV pipeline against human-derived tumors. RESULTS: In agreement with previous bodies of evidence, immunogenicity was driven by predicted affinity. A strong poly-functional and poly-specific immune response was observed with high affinity neoantigens. However, only a high poly-specific vaccine vector was able to completely protect mice from subsequent tumor challenge. More importantly, this pipeline - from the selection of neoantigens to vaccine design - applied to a new model of patient derived tumor xenograft resulted in therapeutic treatment. CONCLUSIONS: These results suggest a feasible strategy for a neoantigen cancer vaccine that is simple and applicable for clinical developments.

The non-inflammatory role of C1q during Her2/neu-driven mammary carcinogenesis.

There is an ever increasing amount of evidence to support the hypothesis that complement C1q, the first component of the classical complement pathway, is involved in the regulation of cancer growth, in addition to its role in fighting infections. It has been demonstrated that C1q is expressed in the microenvironment of various types of human tumors, including breast adenocarcinomas. This study compares carcinogenesis progression in C1q deficient (neuT-C1KO) and C1q competent neuT mice in order to investigate the role of C1q in mammary carcinogenesis. Significantly accelerated autochthonous neu+ carcinoma progression was paralleled by accelerated spontaneous lung metastases occurrence in C1q deficient mice. Surprisingly, this effect was not caused by differences in the tumor-infiltrating cells or in the activation of the complement classical pathway, since neuT-C1KO mice did not display a reduction in C3 fragment deposition at the tumor site. By contrast, a significant higher number of intratumor blood vessels and a decrease in the activation of the tumor suppressor WW domain containing oxidoreductase (WWOX) were observed in tumors from neuT-C1KO as compare with neuT mice. In parallel, an increase in Her2/neu expression was observed on the membrane of tumor cells. Taken together, our findings suggest that C1q plays a direct role both on halting tumor angiogenesis and on inducing apoptosis in mammary cancer cells by coordinating the signal transduction pathways linked to WWOX and, furthermore, highlight the role of C1q in mammary tumor immune surveillance regardless of complement system activation.

Microenvironment, oncoantigens, and antitumor vaccination: lessons learned from BALB-neuT mice.

The tyrosine kinase human epidermal growth factor receptor 2 (HER2) gene is amplified in approximately 20% of human breast cancers and is associated with an aggressive clinical course and the early development of metastasis. Its crucial role in tumor growth and progression makes HER2 a prototypic oncoantigen, the targeting of which may be critical for the development of effective anticancer therapies. The setup of anti-HER2 targeting strategies has revolutionized the clinical outcome of HER2(+) breast cancer. However, their initial success has been overshadowed by the onset of pharmacological resistance that renders them ineffective. Since the tumor microenvironment (TME) plays a crucial role in drug resistance, the design of more effective anticancer therapies should depend on the targeting of both cancer cells and their TME as a whole. In this review, starting from the successful know-how obtained with a HER2(+) mouse model of mammary carcinogenesis, the BALB-neuT mice, we discuss the role of TME in mammary tumor development. Indeed, a deeper knowledge of antigens critical for cancer outbreak and progression and of the mechanisms that regulate the interplay between cancer and stromal cell populations could advise promising ways for the development of the best anticancer strategy.

Multiple roles of perforin in hampering ERBB-2 (Her-2/neu) carcinogenesis in transgenic male mice.

Perforin (pfp)-mediated cytotoxicity is one of the principal immunosurveillance mechanisms involved in the fight against cancer. However, its importance in spontaneous epithelial cancer is still poorly defined. In this study, we use a realistic mouse model that displays many features that are equivalent to human pathology to evaluate the role of pfp-dependent immunosurveillance by comparing tumor progression in rat ERBB-2 (neu) transgenic, pfp-proficient (neu(+)/pfp(+)) or pfp-deficient (neu(+)/pfp(-)) BALB/c male mice. Adult neu(+)/pfp(+) males developed poorly differentiated salivary carcinomas, whereas neu(+)/pfp(-) males displayed their salivary carcinomas noticeably earlier and showed zones of more highly differentiated tumor, indicating that pfp-mediated immunosurveillance is able not only to delay the growth kinetic of an aggressive epithelial tumor, but also to shape its histology. The role of pfp-mediated immunosurveillance appeared to be of even more dramatic importance against the less aggressive male mammary carcinomas. In neu(+)/pfp(+) males, the incidence of mammary carcinomas was a sporadic and late event. In contrast, in neu(+)/pfp(-) males their incidence was four-fold higher. This higher cancer incidence was associated with a 2-fold higher occurrence of persisting mammary remnants, a major risk factor for mammary cancer in male mice, and one that would appear to be due to pfp's previously unidentified involvement in male mammary gland rejection during embryogenesis. This work thus provides further proof of the complex role that the immune system plays in the body and gives new insight into the pathogenesis of epithelial tumors, demonstrating that the penetrance and malignancy of a tumor may be dramatically affected by pfp-dependent mechanisms.

Early onset and enhanced growth of autochthonous mammary carcinomas in C3-deficient Her2/neu transgenic mice.

Aside from its classical role in fighting infections, complement is an important, although poorly understood, component of the tumor microenvironment. In particular, the tumor growth-regulatory activities of complement remain under debate. To assess the role of the complement system in the progression of autochthonous mammary carcinomas, we have crossed complement component 3 (C3)-deficient (C3-/- ) BALB/c male mice with BALB/c females expressing the activated rat Her2/neu oncogene (neuT). Although neuT transgenic mice develop spontaneous mammary cancers with 100% penetrance, a significantly shorter tumor latency (i.e., earlier onset of the first palpable tumor), a higher frequency of multiple tumors (multiplicity), and a dramatic increase in the tumor growth rate were found in neuT-C3-/- animals. The accelerated tumor onset observed in neuT-C3-/- mice was paralleled by an earlier onset of spontaneous lung metastases and by an increase in Her2 expression levels, primarily on the surface of tumor cells. The percentage of immune cells infiltrating neuT carcinomas was similar in C3-deficient and C3-proficient mice, with the exception of a significant increase in the frequency of regulatory T cells in neuT-C3-/- tumors. Of particular interest, the enhanced immunosuppression imparted by C3 deficiency clearly influenced the immunogenic phenotype of autochthonous mammary tumors as neuT-C3-/- malignant cells transplanted into syngeneic immunocompetent hosts gave rise to lesions with a significantly delayed kinetics and reduced incidence as compared with cells obtained from neuT C3-proficient tumors. Finally, increased blood vessel permeability was evident in neuT-C3-/- tumors, although a similar number of tumor vessels was found in neuT and neuT-C3-/- lesions. Altogether, these data suggest that complement plays a crucial role in the immunosurveillance and, possibly, the immunoediting of Her2-driven autochthonous mammary tumors.

Chimeric DNA Vaccines against ErbB2+ Carcinomas: From Mice to Humans.

DNA vaccination exploits a relatively simple and flexible technique to generate an immune response against microbial and tumor-associated antigens (TAAs). Its effectiveness is enhanced by the application of an electrical shock in the area of plasmid injection (electroporation). In our studies we exploited a sophisticated electroporation device approved for clinical use (Cliniporator, IGEA, Carpi, Italy). As the target antigen is an additional factor that dramatically modulates the efficacy of a vaccine, we selected ErbB2 receptor as a target since it is an ideal oncoantigen. It is overexpressed on the cell membrane by several carcinomas for which it plays an essential role in driving their progression. Most oncoantigens are self-tolerated molecules. To circumvent immune tolerance we generated two plasmids (RHuT and HuRT) coding for chimeric rat/human ErbB2 proteins. Their immunogenicity was compared in wild type mice naturally tolerant for mouse ErbB2, and in transgenic mice that are also tolerant for rat or human ErbB2. In several of these mice, RHuT and HuRT elicited a stronger anti-tumor response than plasmids coding for fully human or fully rat ErbB2. The ability of heterologous moiety to blunt immune tolerance could be exploited to elicit a significant immune response in patients. A clinical trial to delay the recurrence of ErbB2+ carcinomas of the oral cavity, oropharynx and hypopharynx is awaiting the approval of the Italian authorities.