murine fibrosarcoma cell line  (ATCC)

Journal: Journal for Immunotherapy of Cancer

Article Title: Novel non-terminal tumor sampling procedure using fine needle aspiration supports immuno-oncology biomarker discovery in preclinical mouse models

doi: 10.1136/jitc-2021-002894

Figure Lengend Snippet: Tumor biopsy using fine needle aspiration (FNA) does not impact tumor growth or welfare. (A) Non-terminal tumor sampling using FNA can both reduce required animal numbers and enable direct correlation of tumor data with treatment outcome by using a single cohort of mice for both tumor biopsying and growth analysis. (B) Subcutaneous flank tumor-bearing mice are anesthetized and the tumor is biopsied at five sites with a 25-gauge needle, collecting the aspirate into cold media and pooling the five aspirates into a single Eppendorf tube. (C and D) Mice were subcutaneously implanted with CT26 tumors on the flank and the tumors were biopsied using FNA either 13 or 20 days later. Tumor growth over time (C) was compared between groups using a mixed effects model and time to endpoint (D) was compared between groups using a log-rank test. No significant differences were seen between FNA-biopsied and control groups. Error bars indicate the mean±SEM. Fourteen mice per group. The results represent one of two independent experiments. (E and F) Mice were subcutaneously implanted with MCA205 tumors on the flank and the tumors were biopsied using FNA 18 days later. Tumor growth over time (E) was compared between groups using a mixed effects model and time to endpoint (F) was compared between groups using a log-rank test. No significant differences were seen between FNA-biopsied and control groups. Error bars indicate the mean±SEM. Eight to 10 mice per group. The results include data from two independent experiments.

Article Snippet: The murine fibrosarcoma cell line MCA205 was obtained from Agonox and maintained in RPMI 1640 media supplemented with 10% FBS.

Techniques: Sampling, Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Novel non-terminal tumor sampling procedure using fine needle aspiration supports immuno-oncology biomarker discovery in preclinical mouse models

doi: 10.1136/jitc-2021-002894

Figure Lengend Snippet: Fine needle aspiration (FNA) can be used to extract cells for flow cytometry at a range of tumor volumes and are representative of the whole tumor. Tumor-bearing mice were sacrificed and the tumors were biopsied using FNA before harvesting the whole tumor (whole tumor digest (WTD)). FNA and WTD samples were analyzed by flow cytometry to determine frequencies of tumor-infiltrating immune cells. (A) The optimal total CD45 + cell count in FNA samples was investigated by plotting the frequencies tumor-infiltrating immune cells (in this example the frequency of FoxP3 − CD4 + T cells) obtained within matched FNA (on the x axis) and WTD (on the y axis) samples from the same tumor. These were separated based on the total number of CD45 + cells within the FNA samples: <1000 CD45 + cells (left) and >1000 CD45 + cells (right). Linear regression was used to calculate the correlation between the frequency obtained in FNA and WTD samples, indicated by the colored lines, and compared with the slope of the black line (representing a slope of 1 with identical frequencies between FNA and WTD samples). Above CD45 + cell counts of 1000 cells, the slope (m) is closer to 1, indicating increased similarity between frequencies in FNA and WTD samples. Data pooled for five tumor models (A20, CT26, MCA205, RENCA and 4T1), 10–34 mice per group. (B) FNA biopsies were collected from mice bearing either CT26 or A20 tumors and analyzed by flow cytometry to determine the total number of CD45 + tumor-infiltrating immune cells within the sample. This was plotted against the volume of the tumor on the day of FNA biopsying for each mouse and linear regression was used to calculate the correlation between the CD45 + cell count and tumor volume for both tumor models, indicated by the solid lines. One hundred twenty-four to 127 mice per group. (C) Using the data from (B), the probability of obtaining >1000 CD45 + cells within an FNA sample was calculated according to the tumor volume for both the CT26 and A20 models. (D–H) The ratio of immune cell frequencies in FNA compared with WTD samples was calculated for (D) CT26, (E) MCA205, (F) A20, (G) 4T1 and (H) RENCA tumors. A ratio close to 1 indicates similar frequencies of cells within FNA and WTD samples. Error bars indicate the mean±SEM. Four to 10 mice per group.

Article Snippet: The murine fibrosarcoma cell line MCA205 was obtained from Agonox and maintained in RPMI 1640 media supplemented with 10% FBS.

Techniques: Flow Cytometry, Cell Counting