The aim of the present work is the set up and characterization of a Natural Killer (NK) cell-sensitive tumour growth model in mice where therapeutic potential of NK cells can be verified. Female C57BL/6 mice (7 weeks of age; 10 animals/group) were used. Green Fluorescent Protein (GFP)-expressing RMA clone B8 (RMA-B8) and RMA-S cells were subcutaneously inoculated into the right flank of anaesthetized animals to assess the lymphoma tumor growth related to the cell variant used. In order to evaluate the effect of contemporaneous inoculum of RMA-B8 and RMA-S cells on tumorigenesis, tumor growth was assessed following inoculum of three mixtures with different concentrations: RMA-B8 104 + RMA-S 105 (Mix-1), RMA-B8104 + RMA-S 106 (Mix-2) and RMA-B8 104 + RMA-S 107 (Mix-3). Inocula of B8-RMA 104 or RMA-S 106 cells alone were used as positive control. Animals were checked for palpable tumour presence and general clinical conditions up to day 12, when they were sacrificed. Only animals that received RMA- B8 cells were followed for tumor appearance up to day 19. Body weight was assessed and, when possible, tumour length (l) and width (w) were measured by callipers at specific check times. Tumour volume was calculated according to the formula v = (l x w2)/2. After inoculation of RMA-B8 cells, up to day 12 animals did not develop tumors. However, starting from day 14 animals injected with RMA-B8 cells showed palpable tumor presence with 100% of mice with tumors by day 19 post-inoculum. When animals were injected with RMA-S 106, inoculation produced tumor in 100% of mice by day 9. When mixtures were tested, animals injected with Mix 1 (104 RMA-B8 + 105 RMA-S cells) showed tumorigenesis with 100% of mice with tumors at day 12, resulting in an anticipated palpable tumors appearance with regards to animals inoculated with cells given alone. A similar result was obtained with the other tested mixtures (104 RMA-B8 + 106 RMA-S and 104 RMA-B8 + 107 RMA-S cells). In order to explain the tumour growth data, we analysed the cellular composition of Mix 1 before inoculum and in the tumour explant at day 12. We observed that whereas in the inoculum, as expected, the GFP-positive RMA-B8 cells were about 1/10 of RMA-S cells, at day 12 the percentage of GFP-positive RMA-B8 cells grew to about 30%. The analysis performed with an MHC-H2 selective monoclonal antibody confirmed that the GFP-positive cells were the MHC-H2 positive cells whereas the GFP-negative cells did not bear the MHC-H2 histocompatibility antigens. The data suggest that following injection of mixed RMA-B8 and RMA-S cells, a shift in the growth rate of the 2 cell lines occurs thus determining an increase in the tumour growth rate. Further studies will be necessary to identify the involved molecular mechanisms.
Set up of a NK cell-sensitive tumour model in C57BL/6 mice
GAMBARI, Roberto;BREVEGLIERI, Giulia;
2009
Abstract
The aim of the present work is the set up and characterization of a Natural Killer (NK) cell-sensitive tumour growth model in mice where therapeutic potential of NK cells can be verified. Female C57BL/6 mice (7 weeks of age; 10 animals/group) were used. Green Fluorescent Protein (GFP)-expressing RMA clone B8 (RMA-B8) and RMA-S cells were subcutaneously inoculated into the right flank of anaesthetized animals to assess the lymphoma tumor growth related to the cell variant used. In order to evaluate the effect of contemporaneous inoculum of RMA-B8 and RMA-S cells on tumorigenesis, tumor growth was assessed following inoculum of three mixtures with different concentrations: RMA-B8 104 + RMA-S 105 (Mix-1), RMA-B8104 + RMA-S 106 (Mix-2) and RMA-B8 104 + RMA-S 107 (Mix-3). Inocula of B8-RMA 104 or RMA-S 106 cells alone were used as positive control. Animals were checked for palpable tumour presence and general clinical conditions up to day 12, when they were sacrificed. Only animals that received RMA- B8 cells were followed for tumor appearance up to day 19. Body weight was assessed and, when possible, tumour length (l) and width (w) were measured by callipers at specific check times. Tumour volume was calculated according to the formula v = (l x w2)/2. After inoculation of RMA-B8 cells, up to day 12 animals did not develop tumors. However, starting from day 14 animals injected with RMA-B8 cells showed palpable tumor presence with 100% of mice with tumors by day 19 post-inoculum. When animals were injected with RMA-S 106, inoculation produced tumor in 100% of mice by day 9. When mixtures were tested, animals injected with Mix 1 (104 RMA-B8 + 105 RMA-S cells) showed tumorigenesis with 100% of mice with tumors at day 12, resulting in an anticipated palpable tumors appearance with regards to animals inoculated with cells given alone. A similar result was obtained with the other tested mixtures (104 RMA-B8 + 106 RMA-S and 104 RMA-B8 + 107 RMA-S cells). In order to explain the tumour growth data, we analysed the cellular composition of Mix 1 before inoculum and in the tumour explant at day 12. We observed that whereas in the inoculum, as expected, the GFP-positive RMA-B8 cells were about 1/10 of RMA-S cells, at day 12 the percentage of GFP-positive RMA-B8 cells grew to about 30%. The analysis performed with an MHC-H2 selective monoclonal antibody confirmed that the GFP-positive cells were the MHC-H2 positive cells whereas the GFP-negative cells did not bear the MHC-H2 histocompatibility antigens. The data suggest that following injection of mixed RMA-B8 and RMA-S cells, a shift in the growth rate of the 2 cell lines occurs thus determining an increase in the tumour growth rate. Further studies will be necessary to identify the involved molecular mechanisms.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
