Limits...
Tumor associated stromal cells play a critical role on the outcome of the oncolytic efficacy of conditionally replicative adenoviruses.

Lopez MV, Viale DL, Cafferata EG, Bravo AI, Carbone C, Gould D, Chernajovsky Y, Podhajcer OL - PLoS ONE (2008)

Bottom Line: Since tumor growth is essentially the result of a continuous cross-talk between malignant and tumor-associated stromal cells, targeting both cell compartments may profoundly influence viral efficacy.These CRAds, expressing or not the Herpes Simplex thymidine kinase gene (Ad-F512 and Ad(I)-F512-TK, respectively) exerted a lytic effect on a panel of human melanoma cells expressing SPARC; but they were completely attenuated in normal cells of different origins, including fresh melanocytes, regardless of whether cells expressed or not SPARC.The whole data indicate that stromal cells might play an important role on the outcome of the oncolytic efficacy of conditionally replicative adenoviruses.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Cellular Therapy, Instituto Leloir, IIBBA-CONICET, Ciudad Autónoma de Buenos Aires, Buenos Aires, Argentina.

ABSTRACT
The clinical efficacy of conditionally replicative oncolytic adenoviruses (CRAd) is still limited by the inefficient infection of the tumor mass. Since tumor growth is essentially the result of a continuous cross-talk between malignant and tumor-associated stromal cells, targeting both cell compartments may profoundly influence viral efficacy. Therefore, we developed SPARC promoter-based CRAds since the SPARC gene is expressed both in malignant cells and in tumor-associated stromal cells. These CRAds, expressing or not the Herpes Simplex thymidine kinase gene (Ad-F512 and Ad(I)-F512-TK, respectively) exerted a lytic effect on a panel of human melanoma cells expressing SPARC; but they were completely attenuated in normal cells of different origins, including fresh melanocytes, regardless of whether cells expressed or not SPARC. Interestingly, both CRAds displayed cytotoxic activity on SPARC positive-transformed human microendothelial HMEC-1 cells and WI-38 fetal fibroblasts. Both CRAds were therapeutically effective on SPARC positive-human melanoma tumors growing in nude mice but exhibited restricted efficacy in the presence of co-administered HMEC-1 or WI-38 cells. Conversely, co-administration of HMEC-1 cells enhanced the oncolytic efficacy of Ad(I)-F512-TK on SPARC-negative MIA PaCa-2 pancreatic cancer cells in vivo. Moreover, conditioned media produced by stromal cells pre-infected with the CRAds enhanced the in vitro viral oncolytic activity on pancreatic cancer cells, but not on melanoma cells. The whole data indicate that stromal cells might play an important role on the outcome of the oncolytic efficacy of conditionally replicative adenoviruses.

Show MeSH

Related in: MedlinePlus

Luciferase activity driven by different variants of SPARC promoter.(A) Left: the different variants of SPARC promoter. Right: Luciferase activity of the different variants of SPARC promoter different cells lines. Data are shown relative to SV40 luciferase activity. Error bars represent mean±SD. (B) Schematic structure of Ad-F512 and Ad(I)-F512-TK genomes.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2663040&req=5

pone-0005119-g001: Luciferase activity driven by different variants of SPARC promoter.(A) Left: the different variants of SPARC promoter. Right: Luciferase activity of the different variants of SPARC promoter different cells lines. Data are shown relative to SV40 luciferase activity. Error bars represent mean±SD. (B) Schematic structure of Ad-F512 and Ad(I)-F512-TK genomes.

Mentions: In order to target both the tumor and stroma compartment we designed a CRAd based on the SPARC promoter since SPARC was shown to be expressed both in malignant and tumor-associated stromal cells [16]. We first assessed the activity of different promoter fragments that were generated maintaining the integrity of specific motives such as two GGA boxes that confer maximal activity, a TATA-like box, two transcription initiation sites [35] and a putative downstream promoter element (DPE [39]). Promoter activity was assessed by cloning each fragment into the promoterless firefly luciferase reporter plasmid pGL3-Basic followed by cell transfection and luminescence quantification. By comparing luciferase levels in A375N melanoma cells that overexpress SPARC compared to breast cancer T-47D and cervical cancer HeLa cell lines that exhibited very low levels of SPARC (see Table 1 for relative SPARC mRNA levels in different cell lines), we selected the −513/+35 fragment, and named it F512Pr (Figure 1A). F512Pr showed 3.3-fold higher activity than the SV40 promoter in A375N cells, while in HeLa and T-47D has similar or less activity than the SV40 promoter (Figure 1A). F512Pr exhibited the highest luciferase activity (1.7 to 4.9 - fold induction over SV40 promoter) in melanoma cell lines that do express high SPARC mRNA levels although no strict relationship was observed between SPARC mRNA levels and promoter activity (Figure S1A and Table 1). We were unable to efficiently transfect additional non-malignant stromal cells such as WI-38 fibroblasts and HMEC-1 endothelial cells (data not shown).


Tumor associated stromal cells play a critical role on the outcome of the oncolytic efficacy of conditionally replicative adenoviruses.

Lopez MV, Viale DL, Cafferata EG, Bravo AI, Carbone C, Gould D, Chernajovsky Y, Podhajcer OL - PLoS ONE (2008)

Luciferase activity driven by different variants of SPARC promoter.(A) Left: the different variants of SPARC promoter. Right: Luciferase activity of the different variants of SPARC promoter different cells lines. Data are shown relative to SV40 luciferase activity. Error bars represent mean±SD. (B) Schematic structure of Ad-F512 and Ad(I)-F512-TK genomes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2663040&req=5

pone-0005119-g001: Luciferase activity driven by different variants of SPARC promoter.(A) Left: the different variants of SPARC promoter. Right: Luciferase activity of the different variants of SPARC promoter different cells lines. Data are shown relative to SV40 luciferase activity. Error bars represent mean±SD. (B) Schematic structure of Ad-F512 and Ad(I)-F512-TK genomes.
Mentions: In order to target both the tumor and stroma compartment we designed a CRAd based on the SPARC promoter since SPARC was shown to be expressed both in malignant and tumor-associated stromal cells [16]. We first assessed the activity of different promoter fragments that were generated maintaining the integrity of specific motives such as two GGA boxes that confer maximal activity, a TATA-like box, two transcription initiation sites [35] and a putative downstream promoter element (DPE [39]). Promoter activity was assessed by cloning each fragment into the promoterless firefly luciferase reporter plasmid pGL3-Basic followed by cell transfection and luminescence quantification. By comparing luciferase levels in A375N melanoma cells that overexpress SPARC compared to breast cancer T-47D and cervical cancer HeLa cell lines that exhibited very low levels of SPARC (see Table 1 for relative SPARC mRNA levels in different cell lines), we selected the −513/+35 fragment, and named it F512Pr (Figure 1A). F512Pr showed 3.3-fold higher activity than the SV40 promoter in A375N cells, while in HeLa and T-47D has similar or less activity than the SV40 promoter (Figure 1A). F512Pr exhibited the highest luciferase activity (1.7 to 4.9 - fold induction over SV40 promoter) in melanoma cell lines that do express high SPARC mRNA levels although no strict relationship was observed between SPARC mRNA levels and promoter activity (Figure S1A and Table 1). We were unable to efficiently transfect additional non-malignant stromal cells such as WI-38 fibroblasts and HMEC-1 endothelial cells (data not shown).

Bottom Line: Since tumor growth is essentially the result of a continuous cross-talk between malignant and tumor-associated stromal cells, targeting both cell compartments may profoundly influence viral efficacy.These CRAds, expressing or not the Herpes Simplex thymidine kinase gene (Ad-F512 and Ad(I)-F512-TK, respectively) exerted a lytic effect on a panel of human melanoma cells expressing SPARC; but they were completely attenuated in normal cells of different origins, including fresh melanocytes, regardless of whether cells expressed or not SPARC.The whole data indicate that stromal cells might play an important role on the outcome of the oncolytic efficacy of conditionally replicative adenoviruses.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Cellular Therapy, Instituto Leloir, IIBBA-CONICET, Ciudad Autónoma de Buenos Aires, Buenos Aires, Argentina.

ABSTRACT
The clinical efficacy of conditionally replicative oncolytic adenoviruses (CRAd) is still limited by the inefficient infection of the tumor mass. Since tumor growth is essentially the result of a continuous cross-talk between malignant and tumor-associated stromal cells, targeting both cell compartments may profoundly influence viral efficacy. Therefore, we developed SPARC promoter-based CRAds since the SPARC gene is expressed both in malignant cells and in tumor-associated stromal cells. These CRAds, expressing or not the Herpes Simplex thymidine kinase gene (Ad-F512 and Ad(I)-F512-TK, respectively) exerted a lytic effect on a panel of human melanoma cells expressing SPARC; but they were completely attenuated in normal cells of different origins, including fresh melanocytes, regardless of whether cells expressed or not SPARC. Interestingly, both CRAds displayed cytotoxic activity on SPARC positive-transformed human microendothelial HMEC-1 cells and WI-38 fetal fibroblasts. Both CRAds were therapeutically effective on SPARC positive-human melanoma tumors growing in nude mice but exhibited restricted efficacy in the presence of co-administered HMEC-1 or WI-38 cells. Conversely, co-administration of HMEC-1 cells enhanced the oncolytic efficacy of Ad(I)-F512-TK on SPARC-negative MIA PaCa-2 pancreatic cancer cells in vivo. Moreover, conditioned media produced by stromal cells pre-infected with the CRAds enhanced the in vitro viral oncolytic activity on pancreatic cancer cells, but not on melanoma cells. The whole data indicate that stromal cells might play an important role on the outcome of the oncolytic efficacy of conditionally replicative adenoviruses.

Show MeSH
Related in: MedlinePlus