Functional roles of pro-differentiation-related proteins in oral cancer progression.
1.Purpose:
We previously reported a tumor suppressive function for S100A14 in oral squamous cell carcinoma (OSCC) (Sapkota et al, 2009; Sapkota et al, 2010). In vitro studies using OSCC-derived cells showed that over-expression S100A14 induced expression of a number of pro-differentiation proteins (such as CK4, CK10, CK13, Involucrin, etc. Preliminary results of ongoing work indicated that higher expression of pro-differentiation proteins in OSCC specimens correlated with better patient survival. Based on these results, we hypothesized that the pro-differentiation proteins have important functional roles in OSCC progression. To answer these questions, one oral premalignant cell line (DOK) and two OSCC-derived (among others SCC25 and H357) cell lines with retroviral mediated knock-down/ overexpression of selected differentiation-related proteins will be injected in the tongue of NOD/SCID mice. The developed tumor xenografts will be harvested after euthanizing the mice and will be analyzed using histology/immunohistological, DNA and mRNA techniques.
2.Distress:
Proper and adequate measures will be followed to minimize distressful situation under injection and tumor measurement. For example, injection of cells (DOK and cancer cells) and measurement of the tumor volume will be done under proper anesthesia with topical analgesia and postoperative care. Mice will not be kept alone in cages. NOD/SCID mice will be kept separately from other animal strains and precautions will be taken to avoid infections due to immunosuppression.
3.Expected benefit:
The planned experiments are expected to uncover the functional roles of selected differentiation-related proteins in OSCC progression. These findings are expected not only to contribute in clarifying the biology of OSCC progression.
4.Number of animals, and what kind:
In this project, we plan to inject DOK, and 2 OSCC-cell lines (SCC25 and H357) with overexpression/knock-down of 3 differentiation-related proteins and their corresponding controls (i,e, 3 different cell-lines with two experimental conditions/one protein: in total 18 different experimental groups for three proteins) in tounge of NOD/SCID mice. We want to use 10 mice in each group (in total 18x10 = 180 mice).
5.How to adhere to 3R
Replacement:
The current study is aimed to investigate the relevance of in vitro findings (already performed) using an in vivo method. The results obtained from in vivo method using SCID mice (in this study) will not only validate/reject in vitro findings but also open the door for the potential clinical application of the proteins examined in oral cancer diagnosis and treatment.
Reduction:
Use of green fluoroscent protein (GFP) labelled cells will enable us to precisely detect tumors and metastases in mice with an IVIS spectrum CT. Hence, use of 10 mice/group will allow sufficient measurements to perform statistical analysis with adequate power.
Refinement:
Tongue injection will cause less tissue damage as compared to other implantation methods. The animals will receive preoperative topical analgesia and as well as postoperative injectable analgesia, and the procedure will be performed under isofluran anesthesia. To ensure consistent disease tracking, all animals will be monitored by the same person. Animals will be reared following standard protocols, and ethically euthanized before they exceed the humane endpoints.
We previously reported a tumor suppressive function for S100A14 in oral squamous cell carcinoma (OSCC) (Sapkota et al, 2009; Sapkota et al, 2010). In vitro studies using OSCC-derived cells showed that over-expression S100A14 induced expression of a number of pro-differentiation proteins (such as CK4, CK10, CK13, Involucrin, etc. Preliminary results of ongoing work indicated that higher expression of pro-differentiation proteins in OSCC specimens correlated with better patient survival. Based on these results, we hypothesized that the pro-differentiation proteins have important functional roles in OSCC progression. To answer these questions, one oral premalignant cell line (DOK) and two OSCC-derived (among others SCC25 and H357) cell lines with retroviral mediated knock-down/ overexpression of selected differentiation-related proteins will be injected in the tongue of NOD/SCID mice. The developed tumor xenografts will be harvested after euthanizing the mice and will be analyzed using histology/immunohistological, DNA and mRNA techniques.
2.Distress:
Proper and adequate measures will be followed to minimize distressful situation under injection and tumor measurement. For example, injection of cells (DOK and cancer cells) and measurement of the tumor volume will be done under proper anesthesia with topical analgesia and postoperative care. Mice will not be kept alone in cages. NOD/SCID mice will be kept separately from other animal strains and precautions will be taken to avoid infections due to immunosuppression.
3.Expected benefit:
The planned experiments are expected to uncover the functional roles of selected differentiation-related proteins in OSCC progression. These findings are expected not only to contribute in clarifying the biology of OSCC progression.
4.Number of animals, and what kind:
In this project, we plan to inject DOK, and 2 OSCC-cell lines (SCC25 and H357) with overexpression/knock-down of 3 differentiation-related proteins and their corresponding controls (i,e, 3 different cell-lines with two experimental conditions/one protein: in total 18 different experimental groups for three proteins) in tounge of NOD/SCID mice. We want to use 10 mice in each group (in total 18x10 = 180 mice).
5.How to adhere to 3R
Replacement:
The current study is aimed to investigate the relevance of in vitro findings (already performed) using an in vivo method. The results obtained from in vivo method using SCID mice (in this study) will not only validate/reject in vitro findings but also open the door for the potential clinical application of the proteins examined in oral cancer diagnosis and treatment.
Reduction:
Use of green fluoroscent protein (GFP) labelled cells will enable us to precisely detect tumors and metastases in mice with an IVIS spectrum CT. Hence, use of 10 mice/group will allow sufficient measurements to perform statistical analysis with adequate power.
Refinement:
Tongue injection will cause less tissue damage as compared to other implantation methods. The animals will receive preoperative topical analgesia and as well as postoperative injectable analgesia, and the procedure will be performed under isofluran anesthesia. To ensure consistent disease tracking, all animals will be monitored by the same person. Animals will be reared following standard protocols, and ethically euthanized before they exceed the humane endpoints.