Perspectives for cancer immunotherapy mediated by p19Arf plus interferon-beta gene transfer

While cancer immunotherapy has gained much deserved attention in recent years, many areas regarding the optimization of such modalities remain unexplored, including the development of novel approaches and the strategic combination of therapies that target multiple aspects of the cancer-immunity cycle. Our own work involves the use of gene transfer technology to promote cell death and immune stimulation. Such immunogenic cell death, mediated by the combined transfer of the alternate reading frame (p14ARF in humans and p19Arf in mice) and the interferon-b cDNA in our case, was shown to promote an antitumor immune response in mouse models of melanoma and lung carcinoma. With these encouraging results, we are now setting out on the road toward translational and preclinical development of our novel immunotherapeutic approach. Here, we outline the perspectives and challenges that we face, including the use of human tumor and immune cells to verify the response seen in mouse models and the incorporation of clinically relevant models, such as patient-derived xenografts and spontaneous tumors in animals. In addition, we seek to combine our immunotherapeutic approach with other treatments, such as chemotherapy or checkpoint blockade, with the goal of reducing dosage and increasing efficacy. The success of any translational research requires the cooperation of a multidisciplinary team of professionals involved in laboratory and clinical research, a relationship that is fostered at the Cancer Institute of Sao Paulo

Evaluation of prognostic markers in breast cancer and functional analysis of CIP4

O câncer de mama é uma doença extremamente heterogênea compreendendo diferentes subtipos moleculares que resultam em evoluções clínicas e condutas terapêuticas distintas. A maior gravidade desta patologia está associada a sua capacidade de formação de metástases Mudanças no padrão de expressão gênica têm sido associadas à manifestação do fenótipo metastático. Neste trabalho, utilizamos microarranjos de tecido (TMAs) para investigar a expressão de 8 biomarcadores candidatos (CIP4, PPIL1, ITGAV, AKAP14, MICA, FXYD1, ARPC3, ABG1) e avaliar seu potencial prognóstico em pacientes com carcinoma ductal invasivo da mama. Destes, ARPC3 PPIL1 e CIP4 mostraram associações estatisticamente significativas com a sobrevida câncer específica e/ou a probabilidade de desenvolvimento de metástases. Determinamos que a expressão aumentada de CIP4 nos tumores está associada a maior probabilidade de desenvolvimento de metástases. CIP4 é uma proteína adaptadora descrita na literatura como moduladora de migração e invasão celular e portanto selecionamos este candidato para caracterização funcional detalhada. Observamos que a expressão de CIP4 encontra-se aumentada em linhagens tumorais com características invasivas. A partir do silenciamento estável e regulado de CIP4 na linhagem metastática MDA-MB-231, determinamos que CIP4 modula positivamente a ativação de MAPK-p38 e a expressão de MMP2 , sugerindo que CIP4 participe em vias de sinalização importantes para a transição epitélio-mesenquima (EMT). O silenciamento de CIP4 resultou em uma redução de aproximadamente 50% da capacidade migratória e invasiva das células tumorais in vitro , e na diminuição da formação de metástases pulmonares in vivo. Coletivamente, nossos resultados indicam que CIP4 tem potencial como marcador de prognóstico assim como um possível alvo terapêutico no controle da disseminação de metástases nos tumores da mama.Breast cancer is an extremely heterogeneous disease comprising different molecular subtypes that result in different clinical outcomes and therapeutic procedures. The severity of this disease is mainly associated with its ability to produce metastasis. Changes in gene expression profile have been associated with the manifestation of the metastatic phenotype. In this study, we used tissue microarrays (TMAs) to investigate the expression of 8 candidate biomarkers (CIP4, PPIL1, ITGAV, AKAP14, MICA, FXYD1, ARPC3 e ABG1) and to evaluate their prognostic potential in patients with invasive ductal breast carcinoma. Among these, ARPC3, PPIL1 and CIP4 showed statistically significant associations with cancer specific survival and/or the patient\'s probability to develop metastasis. We found that increased expression of CIP4 in tumors is associated with a higher probability of developing metastasis. CIP4 is an adaptor protein described in the literature as a modulator of cell migration and invasion and therefore we selected this candidate for detailed functional characterization. We observed that CIP4 expression is increased in tumor cell lines with invasive characteristics. Following the stable and regulated knockdown of CIP4 in the metastatic line MDA-MB-231, we determined that it modulates positively the activation of MAPK-p38 and the expression of MMP2, suggesting that CIP4 participates in important signaling pathways required for the epithelial mesenchymal transition (EMT). CIP4 silencing resulted in an approximate 50% reduction of the migratory and invasive capacity of tumor cells in vitro and decreased the generation of lung metastases in vivo. Collectively, our results indicate that CIP4 has potential as a prognostic marker as well as a potential therapeutic target to control the metastatic dissemination of breast tumors

Evaluation of different immunization systems thats use the oncoprotein E7 of the human Papiloma virus type 16 (HPV 16) genetically fused to the flagellin of the Salmonella enterica sv. muenchen.

O câncer cervical é o segundo maior responsável por mortes atribuídas a câncer em mulheres e dados epidemiológicos tem demonstrado a associação entre a infecção do HPV e o desenvolvimento da neoplasia. Sabe-se que num dado momento da infecção pelo HPV, ocorre integração do genoma viral ao genoma da célula hospedeira e consequente hiperexpressão de dois oncogenes virais, E6 e E7, o que contribui fortemente para a transformação celular. O presente trabalho propõe o uso de vacinas terapêuticas expressando a oncoproteína E7 do HPV-16 geneticamente fusionada à porção amino terminal da flagelina FliCd de Salmonella enterica sv müenchen; e verificação de seu possível papel adjuvante. Vacinas de DNA foram construídas de modo a direcionar as proteínas hibridas ao espaço intracitoplasmático. A verificação da expressão in vitro foi feita utilizando transfecções de culturas celulares, seguida de imunofluorescência e imunodetecção. Em seguida, essas construções vacinais foram administradas em camundongos C57BL6. Ensaios de ELISPOT foram feitos para mensuração o nível de linfócitos T secretores de IFN-g. Os dados de imunofluorescência e imunodetecção demonstraram a correta expressão da proteína. Ensaios de proteção realizados com 5 x 106 células TC-1 administrada por via subcutânea mostraram 80% de proteção em camundongos que haviam recebido previamente 4 doses das vacinas de DNA por biobalística. Ensaios de ELISPOT mostraram em média 9 células do baço secretoras de IFN-g por 106 células do baço (INF-g+/106) responsivas ao peptídeo CD8 / E7 de forma específica. Nossos dados sugerem que a formulação vacinal possui um efeito terapêutico significativo frente ao desafio com as células tumorais TC-1. Em paralelo, foi construída a cepa de salmonela vacinal SL FlaE7 que não mostrou efeito protetor frente ao desafio com células TC-1.The cervical cancer is the second major responsible for deaths attributed to cancer in women and epidemiologic data have been demonstrating the association between the infection of HPV and the development of this illness. It is known that in a certain moment of the infection with HPV, occurs the integration of the viral genome in the genome of the host cell and consequent over expression of two oncogenes, E6 and E7, it strongly contributes to the transformation of that cell. The present work proposes the use of DNA vaccines expressing the oncoprotein E7 of HPV-16 genetically fused to the portion A-terminal of the flagellin FliCd of Salmonella enterica sv müencheun and verification of your possible performance as adjuvant. The DNA vaccines were constructed in expression vector for eukaryotic to address the hybrid proteins to the intracitoplasmatic space. The verification of the expression in vitro was made using transfections of cellular cultures (COS-7) followed by immunofluorescence and western blot analyses. Soon after, those vaccines were injected in mice C57BL6 that were challenged then with 5^105 tumor cells TC-1 subcutaneous. ELISPOT assays were performed for measure the level of spleen cells secreting interferon g. The immunofluorescence and Western blot data are complementary demonstrating the correct expression of the protein. Protection assays showed 80% of protection in mice that had previously received 4 doses of the DNA vaccines in gene gun form. Assays of ELISPOT show 9 cells of the spleen secreting of IFN-g (average) for 106 cells of the spleen (INF-g /106). Our data suggest that the formulation of vaccines possesses a significant therapeutic effect front to the challenge with the tumor cells TC-1 accompanist splenocyte IFN-g secretory

Perspectives for cancer immunotherapy mediated by p19Arf plus interferon-beta gene transfer

While cancer immunotherapy has gained much deserved attention in recent years, many areas regarding the optimization of such modalities remain unexplored, including the development of novel approaches and the strategic combination of therapies that target multiple aspects of the cancer-immunity cycle. Our own work involves the use of gene transfer technology to promote cell death and immune stimulation. Such immunogenic cell death, mediated by the combined transfer of the alternate reading frame (p14ARF in humans and p19Arf in mice) and the interferon-β cDNA in our case, was shown to promote an antitumor immune response in mouse models of melanoma and lung carcinoma. With these encouraging results, we are now setting out on the road toward translational and preclinical development of our novel immunotherapeutic approach. Here, we outline the perspectives and challenges that we face, including the use of human tumor and immune cells to verify the response seen in mouse models and the incorporation of clinically relevant models, such as patient-derived xenografts and spontaneous tumors in animals. In addition, we seek to combine our immunotherapeutic approach with other treatments, such as chemotherapy or checkpoint blockade, with the goal of reducing dosage and increasing efficacy. The success of any translational research requires the cooperation of a multidisciplinary team of professionals involved in laboratory and clinical research, a relationship that is fostered at the Cancer Institute of Sao Paulo

MCF10A, Hs578T and transformed cells characterization.

<p>(A, B) Cell Morphology analysis with phalloidin; (C, D) <i>In vitro</i> cell growth analysis; (E, F) Colony formation assay in semi-solid medium; (G, H) Migration and invasiveness assays. ANOVA test followed by Tukey’s test for post hoc comparison were used for statistical analysis. The results are presentes as mean±SD. Asterisks show statistically significant expression differences (p <0.05). The experiments were performed in three independent experiments. Phalloidin (red), DAPI (blue), and merged images (original magnification, x20).</p

Characterization of CD90 expression in MCF10A and Hs578T and transformed cells.

<p>The expression of CD90 was analysed by qRT-PCR, Western-blot and immunofluorescence microscopy in MCF10A (A,B,C) and Hs578T (D,E,F) cell lines. For Western blot analyses, the cells were lysed and the total protein was separated by SDS-PAGE. The gels were electrotransfered onto nitrocellulose membranes. The membranes were then stripped and reprobed with β-actin antibodies to confirm that equal amounts of each protein extract were loaded. The immunoblots shown are representative of a total of three independent experiments. CD90 (red), DAPI (blue), and merged images (original magnification, x20).</p

In vivo metastasis assay.

<p><i>In vivo</i> assays for metastasis in rat lung with parental and transformed cell lines. Cells (1x10⁶) were injected into tail vein of immunodeficient rats. After 45 days the animals were euthanized and the lungs were analyzed. (A) Lungs and H&E lung sections of animals injected with the MCF10A, MCF10A-empty vector and MCF10A/CD90+ cell lines. (B) Lungs and H&E lung sections of animals injected with the Hs578T, Hs578T non-target and Hs578T/shCD90 cell lines. C) Number of lesions on the surface of the lungs of the animals injected with the cells of the strains MCF10A, MCF10A-empty vector and MCF10A/CD90+. (D) Number of lesions on the surface of the lungs of the animals injected with the cells of the strains Hs578T, Hs578T non-target and Hs578T/shCD90. ANOVA test followed by Tukey’s test for post hoc comparison were used for statistical analysis. Asterisks show statistically significant expression differences (p <0.05). The experiments were performed in two independent experiments in triplicate.</p

Kaplan-Meier survival analysis.

<p>(A) Kaplan-Meier (KM) survival curves at the 120-month follow up point (TFU). (B) Kaplan-Meier metastasis-free survival (MFS) curves at the 120-month follow up point. For both TFU and MFS KM survival curves, the median of the CD90 H-score was used to discriminate patients with high CD90 expression (red line) and low CD90 expression (blue line). P-values were obtained by log-rank tests.</p

Analysis of the activity of the responsive element of the transcription factor AP-1.

<p>MCF10A, MCF10A-empty vector and MCF10A/CD90+ cells (2x10⁵) were plated and transfected with vector pGL4.44 and pRL-TK, deprived for FCS and EGF or treated with EGF. The reading of luciferase activity was done after 48 hours of transfection. The luciferase activity was normalized by the activity/constitutive expression of Renilla. ANOVA test followed by Tukey’s test for post hoc comparison were used for statistical analysis. Asterisks show statistically significant expression differences (p <0.05). The experiments were performed in three independent experiments.</p

Immunohistochemistry analysis of human Anti-CD90/Thy1 of the rats lungs.

<p>Immunohistochemical staining for human Anti-CD90/Thy1 was peformed on formalin-fixed, paraffin embedded 3μm section of lungs tissues from rats injected with (A) MCF10A, MCF10A-empty vector, MCF10A/CD90+ cell lines and (B) Hs578T, Hs578T non-target and Hs578T/shCD90 cell lines. Original magnification, x40.</p