RESUMO
Rationale: The tumor microenvironment (TME) and its multifaceted interactions with cancer cells are major targets for cancer treatment. Single-cell technologies have brought major insights into the TME, but the resulting complexity often precludes conclusions on function. Methods: We combined single-cell RNA sequencing and spatial transcriptomic data to explore the relationship between different cancer-associated fibroblast (CAF) populations and immune cell exclusion in breast tumors. The significance of the findings was then evaluated in a cohort of tumors (N=75) from breast cancer patients using immunohistochemistry analysis. Results: Our data show for the first time the degree of spatial organization of different CAF populations in breast cancer. We found that IL-iCAFs, Detox-iCAFs, and IFNγ-iCAFs tended to cluster together, while Wound-myCAFs, TGFß-myCAFs, and ECM-myCAFs formed another group that overlapped with elevated TGF-ß signaling. Differential gene expression analysis of areas with CD8+ T-cell infiltration/exclusion within the TGF-ß signaling-rich zones identified elastin microfibrillar interface protein 1 (EMILIN1) as a top modulated gene. EMILIN1, a TGF-ß inhibitor, was upregulated in IFNγ-iCAFs directly modulating TGFß immunosuppressive function. Histological analysis of 75 breast cancer samples confirmed that high EMILIN1 expression in the tumor margins was related to high CD8+ T-cell infiltration, consistent with our spatial gene expression analysis. High EMILIN1 expression was also associated with better prognosis of patients with breast cancer, underscoring its functional significance for the recruitment of cytotoxic T cells into the tumor area. Conclusion: Our data show that correlating TGF-ß signaling to a CAF subpopulation is not enough because proteins with TGF-ß-modulating activity originating from other CAF subpopulations can alter its activity. Therefore, therapeutic targeting should remain focused on biological processes rather than on specific CAF subtypes.
Assuntos
Neoplasias da Mama , Fibroblastos Associados a Câncer , Feminino , Humanos , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral , Glicoproteínas de Membrana/metabolismoRESUMO
The study of cellular networks mediated by ligand-receptor interactions has attracted much attention recently owing to single-cell omics. However, rich collections of bulk data accompanied with clinical information exists and continue to be generated with no equivalent in single-cell so far. In parallel, spatial transcriptomic (ST) analyses represent a revolutionary tool in biology. A large number of ST projects rely on multicellular resolution, for instance the Visium™ platform, where several cells are analyzed at each location, thus producing localized bulk data. Here, we describe BulkSignalR, a R package to infer ligand-receptor networks from bulk data. BulkSignalR integrates ligand-receptor interactions with downstream pathways to estimate statistical significance. A range of visualization methods complement the statistics, including functions dedicated to spatial data. We demonstrate BulkSignalR relevance using different datasets, including new Visium liver metastasis ST data, with experimental validation of protein colocalization. A comparison with other ST packages shows the significantly higher quality of BulkSignalR inferences. BulkSignalR can be applied to any species thanks to its built-in generic ortholog mapping functionality.
RESUMO
Hormone therapy (HT) is an effective treatment for metastatic endometrial carcinoma (mEC), with limited toxicity and low cost. We focused on molecular analysis of mECs treated by HT and, for the first time to date, we compared the genomic profiles of paired metastasis and primary ECs. The main objective was to identify predictive factors of the response to HT as well as specific altered signaling pathways driving mEC biology. From 1052 patients with EC treated by HT in two French cancer centers, 32 with endometrioid EC and 6 with high grade serous EC were included. We evaluated hormone receptors (HR) and mismatch repair proteins expression by immunohistochemistry and gene alterations by targeted next-generation sequencing and array-based comparative genomic hybridization. Several variables were tested in univariate and multivariate analyses to identify potential associations with (i) the clinical benefit of HT (CBHT) and (ii) a longer response (>18 months) (LRHT) and overall survival (OS). We compared the biological and genomic profiles of 11 primary/metastatic EC pairs. Thirty tumors (78.9%) were HR-positive and 6 (15.8%) showed microsatellite instability (MSI). The genomic profiles of 34 tumors showed an average altered genome of 3.26%, DNA repair homologous recombination deficiency in five tumors (14.7%), and 17 regions significantly targeted by amplification/deletion. Thirty-three tumors had 273 variants (158 genes, median of 7 mutations/sample), including 112 driver mutations. TP53, PTEN, PPP2R1A, ARID1A, FGFR2, and PIK3CA were the most frequently mutated. Based on the genomic status, nine oncogenic pathways were altered in more than 25% of primary EC. Clinically, 22 (57.9%) and 6 (15.8%) patients presented CBHT and LRHT, respectively. Neither oncogenic pathways alterations nor the variables tested were associated with CBHT and LRHT. Only patient's age, mitotic index and the presence of at least one HR were associated with OS. Paired analysis of the primary/metastatic samples showed that among the 22 mutations acquired in the metastatic counterparts, the most frequently targeted genes were involved in pathways that might confer a selective advantage to cancer metastasis including hormone resistance. In conclusion, only patient's age, mitotic index and the presence of at least one HR were associated with OS. The identification of gene mutations newly acquired in metastasis might help to better understand the formation of EC metastasis and select the best actionable candidates for HT-treated patients at the metastatic stage.
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Cancer stem cells (CSCs) are a small but critical cell population for cancer biology since they display inherent resistance to standard therapies and give rise to metastases. Despite accruing evidence establishing a link between deregulation of epitranscriptome-related players and tumorigenic process, the role of messenger RNA (mRNA) modifications in the regulation of CSC properties remains poorly understood. Here, we show that the cytoplasmic pool of fat mass and obesity-associated protein (FTO) impedes CSC abilities in colorectal cancer through its N6,2'-O-dimethyladenosine (m6Am) demethylase activity. While m6Am is strategically located next to the m7G-mRNA cap, its biological function is not well understood and has not been addressed in cancer. Low FTO expression in patient-derived cell lines elevates m6Am level in mRNA which results in enhanced in vivo tumorigenicity and chemoresistance. Inhibition of the nuclear m6Am methyltransferase, PCIF1/CAPAM, fully reverses this phenotype, stressing the role of m6Am modification in stem-like properties acquisition. FTO-mediated regulation of m6Am marking constitutes a reversible pathway controlling CSC abilities. Altogether, our findings bring to light the first biological function of the m6Am modification and its potential adverse consequences for colorectal cancer management.
Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Neoplasias Colorretais/metabolismo , Citoplasma/metabolismo , Desmetilação , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Metiltransferases/metabolismo , Proteínas Nucleares/metabolismo , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: In luminal androgen receptor (AR) tumours, FOXA1 may direct AR to sites occupied by ER in luminal tumours, thus stimulating proliferation. METHODS: AR and FOXA1 expression were evaluated by immunohistochemistry in 333 non-metastatic triple-negative breast cancers (TNBC). Positivity threshold was set at ≥ 1% staining. Lymphocytic infiltration, PD-L1expression, PIK3CA mutations, PTEN defects and BRCA1 promoter methylation were assessed. RESULTS: AR + /FOXA1 + tumours (42.4%) were more frequently: found in older patients, lobular, of lower nuclear grade, with more frequently PIK3CA mutations; exhibited less frequently BRCA1 promoter methylation, defects of PTEN and PD-L1 expression than others. Recurrence-free and overall survivals were significantly lower for AR + /FOXA1 + TNBC (median follow-up: 7.8 years). CONCLUSIONS: AR + /FOXA1 + expression defines a luminal-like TNBC subgroup affected with a worse outcome compared to other TNBC and a higher risk of late recurrences. This subgroup appears enriched in PIK3CA mutations, suggesting a role for PI3K inhibitors in this subgroup.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Fator 3-alfa Nuclear de Hepatócito/genética , Recidiva Local de Neoplasia/genética , Receptores Androgênicos/genética , Neoplasias de Mama Triplo Negativas/genética , Idoso , Antígeno B7-H1/genética , Proteína BRCA1/genética , Biomarcadores Tumorais/genética , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Pessoa de Meia-Idade , Mutação/genética , Recidiva Local de Neoplasia/classificação , Recidiva Local de Neoplasia/patologia , Prognóstico , Fatores de Risco , Neoplasias de Mama Triplo Negativas/classificação , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The AP-1 transcription factor Fra-1 is aberrantly expressed in a large number of cancers and plays crucial roles in cancer development and progression by stimulating the expression of genes involved in these processes. However, the control of Fra-1 transactivation ability is still unclear and here we hypothesized that PKCθ-induced phosphorylation could be necessary to obtain a fully active Fra-1 protein. Using MCF7 stable cells overexpressing equivalent levels of unphosphorylated Fra-1 or PKCθ-phosphorylated Fra-1, we showed that PKCθ-induced phosphorylation of Fra-1 was crucial for the stimulation of MMP1 and IL6 expression. Consistently, we found a significant positive correlation between PRKCQ (coding for PKCθ) and MMP1 mRNA expression levels in human breast cancer samples. PKCθ-induced phosphorylations, in part at T217 and T227 residues, strongly and specifically increased Fra-1 transcriptional activity through the stimulation of Fra-1 transactivation domain, without affecting JUN factors. More importantly, these phosphorylations were required for Fra-1-induced migration of breast cancer cells and phosphorylated Fra-1 expression was enriched at the invasion front of human breast tumors. Taken together, our findings indicate that PKCθ-induced phosphorylation could be important for the function of Fra-1 in cancer progression.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/enzimologia , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Isoenzimas/genética , Células MCF-7 , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Invasividade Neoplásica , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C-theta , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-fos/genética , Interferência de RNA , Transdução de Sinais , Transcrição Gênica , TransfecçãoRESUMO
Advanced Epithelial Ovarian Cancer (EOC) patients frequently relapse by 24 months and develop resistant disease. Research on EOC therapies relies on cancer cell lines established decades ago making Patient Derived Xenografts (PDX) attractive models, because they are faithful representations of the original tumor. We established 35 ovarian cancer PDXs resulting from the original graft of 77 EOC samples onto immuno-compromised mice. PDXs covered the diversity of EOC histotypes and graft take was correlated with early patient death. Fourteen PDXs were characterized at the genetic and histological levels. PDXs reproduced phenotypic features of the ovarian tumors of origin and conserved the principal characteristics of the original copy number change (CNC) profiles over several passages. However, CNC fluctuations in specific subregions comparing the original tumor and the PDXs indicated the oligoclonal nature of the original tumors. Detailed analysis by CGH, FISH and exome sequencing of one case, for which several tumor nodules were sampled and grafted, revealed that PDXs globally maintained an oligoclonal structure. No overgrowth of a particular subclone present in the original tumor was observed in the PDXs. This suggested that xenotransplantation of ovarian tumors and growth as PDX preserved at least in part the clonal diversity of the original tumor. We believe our data reinforce the potential of PDX as exquisite tools in pre-clinical assays.
Assuntos
Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Xenoenxertos/metabolismo , Neoplasias Ovarianas/genética , Transplante Heterólogo/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células Clonais/metabolismo , Células Clonais/patologia , Feminino , Xenoenxertos/patologia , Humanos , Camundongos Nus , Pessoa de Meia-Idade , Metástase Neoplásica , Recidiva Local de Neoplasia , Neoplasias Ovarianas/patologia , Prognóstico , Análise de SobrevidaRESUMO
The management of pT1a-bN0M0 breast cancer remains an area of controversy. Data from 714 patients classified as having pT1a-bN0M0 breast cancer and treated, from 1999 to 2004 in the Languedoc-Roussillon France, were analyzed. The human epidermal growth factor receptor 2 (HER2) status analyses were centralized. The objective of this study was to describe the prognosis of pT1a-bN0M0 breast cancer according to HER2 distribution and hormonal status. The median follow-up was 6.4 years. Ten-year overall survival was 94%. HER2 overexpression was observed in 6.1% of the patients. The 10-year prognosis of patients with HER2-positive tumors was worse than that of those with HER2-negative (disease-free survival 73% vs. 89%, P < 0.0001). Tumor size (T1a/T1b) was not a relevant prognostic factor. The co-expression of HER2 with hormonal receptors (HR) was associated with high recurrence at 10 years. In both univariate and multivariate analyses, the most relevant prognostic factor for this population was HER2 amplification. In multivariate analysis, patients with HER2-positive tumors had higher risk of mortality (HR, 3.89; 95% CI, 1.58-9.56). In pT1a-bN0M0 breast cancers, HER2 amplification or overexpression is a risk factor for recurrence. In HER2-positive breast cancers, HR expression is associated with a poor prognosis despite the hormone therapy. For this population, a personalized management may be required.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Recidiva Local de Neoplasia/tratamento farmacológico , Receptor ErbB-2/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Quimioterapia Adjuvante , Intervalo Livre de Doença , Feminino , França , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Prognóstico , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Fatores de Risco , Resultado do TratamentoRESUMO
Patient derived xenografts (PDXs) are increasingly appreciated models in cancer research, particularly for preclinical testing, as they reflect the patient's tumor biology more accurately than cancer cell lines. We have established a collection of 20 breast PDXs and characterized their biological and clinical features, as well as their genetic stability. While most PDXs originated from triple negative breast cancers (70%), our collection comprised five ER + cases (25%). Remarkably, the tumors that produced PDXs derived from a subset of aggressive breast cancers with a high proportion of grade 3 tumors and reduced recurrence-free survival. Consistent with this, we found significant differences between the transcriptomic signatures of tumors that produced a PDX (Take) and those that did not (No Take). The PDXs faithfully recapitulate the histological features of their primary tumors, and retain an excellent conservation of molecular classification assignment and Copy Number Change (CNC). Furthermore, the CNC profiles of different PDXs established from the same tumor overlap significantly. However, a small fraction of CNCs in the primary tumor that correspond to oligoclonal events were gradually lost during sequential passaging, suggesting that the PDXs' genetic structure eventually stabilizes around a dominant clone present in the tumor of origin. Finally, de novo occurring genetic events covering up to 9% of the genome were found in only a minority of the PDXs, showing that PDXs have limited genetic instability. These data show that breast cancer PDXs represent a subset of aggressive tumors prone to relapse, and that despite of an excellent conservation of original features, they remain genetically dynamic elements.
Assuntos
Neoplasias da Mama , Regulação Neoplásica da Expressão Gênica , Transcriptoma , Animais , Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Perfilação da Expressão Gênica , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Transplante de NeoplasiasRESUMO
The management of colorectal liver metastases has been improved these last years. The efficacy of chemotherapy regimens and targeted therapies has led to a better prognosis. It has also allowed the resection of metastases initially unresectable. In this setting, the pathologist plays a major role. He is involved in the gross examination, in order to perform an adequate sampling of the lesions. He is also involved at the morphological level, for the assessment of the pathological response, which is now recognized as a prognostic factor and a marker of sensitivity or resistance to a given treatment. Moreover, the determination of predictive markers of response or resistance to induction treatments will constitute a supplementary and major challenge for the pathologist.
Assuntos
Adenocarcinoma/secundário , Antineoplásicos/uso terapêutico , Neoplasias Colorretais/patologia , Quimioterapia de Indução , Neoplasias Hepáticas/secundário , Patologia Clínica , Papel do Médico , Adenocarcinoma/química , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/cirurgia , Antineoplásicos/efeitos adversos , Biomarcadores Tumorais/análise , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Terapia Combinada , Gerenciamento Clínico , Humanos , Comunicação Interdisciplinar , Neoplasias Hepáticas/química , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/cirurgia , Terapia de Alvo Molecular , Gradação de Tumores , Prognóstico , Resultado do TratamentoRESUMO
Despite recent advances in the treatment of colon cancer, tumor resistance is a frequent cause of chemotherapy failure. To better elucidate the molecular mechanisms involved in resistance to irinotecan (and its active metabolite SN38), we established SN38-resistant clones derived from HCT-116 and SW48 cell lines. These clones show various levels (6- to 60-fold) of resistance to SN-38 and display enhanced levels of activated MAPK p38 as compared with the corresponding parental cells. Because four different isoforms of p38 have been described, we then studied the effect of p38 overexpression or downregulation of each isoform on cell sensivity to SN38 and found that both α and ß isoforms are involved in the development of resistance to SN38. In this line, we show that cell treatment with SB202190, which inhibits p38α and p38ß, enhanced the cytotoxic activity of SN38. Moreover, p38 inhibition sensitized tumor cells derived from both SN38-sensitive and -resistant HCT116 cells to irinotecan treatment in xenograft models. Finally, we detected less phosphorylated p38 in primary colon cancer of patients sensitive to irinotecan-based treatment, compared with nonresponder patients. This indicates that enhanced level of phosphorylated p38 could predict the absence of clinical response to irinotecan. Altogether, our results show that the p38 MAPK pathway is involved in irinotecan sensitivity and suggest that phosphorylated p38 expression level could be used as a marker of clinical resistance to irinotecan. They further suggest that targeting the p38 pathway may be a potential strategy to overcome resistance to irinotecan-based chemotherapies in colorectal cancer.
Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/enzimologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Camptotecina/análogos & derivados , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Camptotecina/administração & dosagem , Camptotecina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Fluoruracila/administração & dosagem , Células HCT116 , Humanos , Imidazóis/administração & dosagem , Imidazóis/farmacologia , Imuno-Histoquímica , Irinotecano , Isoenzimas , Leucovorina/administração & dosagem , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Nus , Fosforilação , Piridinas/administração & dosagem , Piridinas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The pathologist's role in 2008 includes diagnosic, prognostic and predictive implications. Histopathologic criteria are still essential to manage the disease of cancer patients. They need to be based on standardized pathology reports and can use ancillary techniques such as immunohistochemistry and molecular biology. However, all the criteria used are not sufficient enough to provide accurate prognostic and predictive parameters for every patient and disease. This is the reason why translational studies more often involve the pathologist in prospective trials. The modern pathology is really integrated in this strategy, in terms of multidisciplinary approach, tumor banking, tissue microarray, molecular biology and new fixatives developments. All these subjects are discussed in this review article.
Assuntos
Neoplasias/patologia , Neoplasias/terapia , Patologia Clínica , Fixadores , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Serviço Hospitalar de Patologia , Prognóstico , Papel (figurativo)RESUMO
INTRODUCTION: An emerging hypothesis suggests that cytokines could play an important role in cancer as potential modulators of angiogenesis and leucocyte infiltration. METHODS: A novel multiplexed flow cytometry technology was used to measure the expression of 17 cytokines (IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 [p70], IL-13, IL-17, granulocyte colony-stimulating factor [CSF], granulocyte-macrophage CSF, IFN-gamma, monocyte chemoattractant protein [MCP]-1, macrophage inflammatory protein [MIP]-1beta, tumour necrosis factor [TNF]-alpha) at the protein level in 105 breast carcinoma. B lymphocyte, T lymphocyte and macrophage levels were determined by immunohistochemistry. RESULTS: Fourteen of the 17 cytokines were expressed in breast carcinoma, whereas only nine cytokines could be detected in normal breast. Most cytokines were more abundant in breast carcinoma than in normal breast, with IL-6, IL-8, granulocyte CSF, IFN-gamma, MCP-1 and MIP-1beta being very abundant. IL-2, IL-6, IL-8, IL-10, IFN-gamma, MCP-1, MIP-1beta and TNF-alpha, and to a lesser extent IL-1beta and IL-13 exhibited levels of expression that were inversely correlated to oestrogen receptor and progesterone receptor status. Most cytokines were not correlated with age at cancer diagnosis, tumour size, histological type, or lymph node status. However, IL-1beta, IL-6, IL-8, IL-10, IL-12, MCP-1 and MIP-1beta were more abundant in high-grade tumours than in low-grade tumours. In addition, IL-8 and MIP-1beta were expressed to a greater degree in HER2-positive than in HER2-negative patients. The expression of most of the studied cytokines was correlated to levels of activator protein-1, which is known to regulate numerous cytokines. Overexpression of MCP-1 and MIP-1beta were linked to B lymphocyte, T lymphocyte and macrophage infiltration, whereas high levels of IL-8 were correlated with high macrophage content in tumour. Moreover, IL-8 positive tumours exhibited increased vascularization. CONCLUSION: We found that multiple cytokines were overexpressed in oestrogen receptor negative breast carcinoma, and that the three major cytokines--MCP-1, MIP-1beta and IL-8--were correlated with inflammatory cell component, which could account for the aggressiveness of these tumours.
Assuntos
Neoplasias da Mama/metabolismo , Citocinas/metabolismo , Neovascularização Patológica , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Estudos de Casos e Controles , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , InflamaçãoRESUMO
Gene therapy for cancer using suicide genes such as the herpes simplex virus thymidine kinase gene (HSVtk) has been explored extensively in preclinical and clinical studies. We have improved the use of HSVtk by combining it with two cytokine genes encoding granulocyte/macrophage-colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2), and determined their additive/synergistic effects on tumor regression and inhibition of metastases in the non-immunogenic, spontaneously metastatic mammary tumor model, 4T1. Two adenoviral vectors (AV) were constructed, one carrying HSVtk (AV-TK) and the second (AV-GM/IL2) carrying Gm-CSf and Il2. Only the combination of AV-TK/GCV and AV-GM/IL2 showed a significant decrease in tumor growth and reduction of distant metastases with 25% of the tumors undergoing complete regression. When surgical excision of primary tumors was included in the regimen, local treatment with AV-TK/GCV plus AV-GM/IL2 further enhanced long-term survival. A fraction of the treated mice developed anti-tumor immunity and survived a second challenge with 4T1. Functional analyses demonstrated infiltration of lymphocytes within the tumor and a strong tumor-specific cytotoxic T lymphocyte response in TK- plus cytokine-treated animals. These data indicate that the coexpression of GM-CSF and IL-2 can augment the effect of HSVtk suicide gene therapy.