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Blood, Vol. 113, Issue 25, 6382-6385, June 18, 2009
Type I natural killer T cells suppress tumors caused by p53 loss in mice
Blood Swann et al.
113: 6382
Supplemental materials for: Swann et al
Histology
Abnormal tissues were fixed in 10% neutral-buffered formalin (NBF) and later processed for hematoxylin and eosin (H&E) staining and examination. Osteosarcomas were decalcified before paraffin embedding. In all cases, paraffin embedding, tissue sectioning, and H&E staining were carried out by the Histology Laboratory, Peter MacCallum Cancer Centre. Tissue sections were analysed by a trained pathologist (Dr. Bill Murray, Pathology Division, Peter MacCallum Cancer Centre) in a blinded fashion. MCA model
Fibrosarcomas were induce by injecting male mice in the hind flank with the desired quantity of 3-methycholanthrene (MCA, Sigma Chemical Company, St Louis, MO, USA) (range 5–100 µg/mouse) diluted in corn oil to a final volume of 100 µL. Due to the viscous nature of the corn oil, injections were performed using a 21-guage needle. Following MCA-injection mice were monitored weekly for tumor growth over a period of 250 days.
Files in this Data Supplement:
- Figure S1. Examples of osteosarcomas derived from p53+∕− mice (JPG, 177 KB)
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Osteosarcomas were harvested from mice, fixed in 10% neutral-buffered formalin (NBF), then de-calcified in 20% EDTA (w/v in H2O) at 4°C for up to 2 weeks. After de-calcification, osteosarcomas were washed in tap water, re-fixed overnight in 10% NBF, then paraffin-embedded, sectioned, and stained with H & E for analysis. (A–C) are examples of osteosarcomas derived from p53+∕− B6 mice, (D–E) are osteosarcomas from p53+∕−Jα18−∕− mice and (G–I) are osteosarcomas from p53+∕−CD1d−∕− mice. Images were taken at ×20 magnification.
- Figure S2. Examples of spindle cell tumors derived from p53+∕− mice (JPG, 174 KB)
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Spindle cell tumors were harvested from mice, fixed in 10% neutral-buffered formalin (NBF), then paraffin-embedded, sectioned, and stained with H & E for analysis. A–C) are examples of spindle cell tumors derived from p53+∕− B6 mice, (D–F) are spindle cell tumors from p53+∕−Jα18−∕− mice and (G–I) are spindle cell tumors from p53+∕−CD1d−∕− mice. Images were taken at ×20 magnification.
- Figure S3. Examples of other tumors derived from p53+∕− mice (JPG, 247 KB)
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(A–C) are examples of hematopoietic tumors, and three cases of large cell lymphoma are depicted. (D–I) are examples of sections from tumors classified as carcinoma/adenocarcinoma: (D–F) are three examples of squamous cell carcinoma, (G) is a lung adenocarcinoma, (H) is an ovarian carcinoma, and (I) is a case of ductal carcinoma in situ. (J–L) are examples of angiosarcomas (which were included in the spindle cell tumors and sarcomas category). (A, D, G, and J) are tumors from p53+∕− B6 mice, (B, E, H, and K) are tumors from p53+∕−Jα18−∕− mice, and (C, F, I, and L) are tumors from p53+∕−CD1d−∕− mice. All tumors were harvested from mice, fixed in 10% neutral-buffered formalin (NBF), then paraffin-embedded, sectioned, and stained with H & E for analysis. Images were taken at ×20 magnification.
- Figure S4. Transplant and immunogenicity of tumors derived from p53+∕−Jα18−∕− mice (JPG, 65.2 KB)
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Four lymphoma lines (designated phJα2, phJα6, phJα12, and phJα98) derived from p53+∕−.Jα18−∕− mice and two lymphomas lines (designated phWT4 and phWT147) derived from p53+∕−.WT mice were transplanted into either WT, Jα18−∕−, or CD1d−∕− hosts. (A) 1 × 105 phJα2 cells were injected i.v. into 4–5 mice per group. Data is from one of two independent experiments. (B) 2 × 106 phJα6 cells and (C) 2 × 106 phJα12 cells were injected i.p. into 4 mice per group. Data is from one experiment. (D) Between 4 × 105 and 1 × 106 phJα98 cells were injected i.p. into 6 mice per group. Data is pooled from two independent experiments. (E) 5 × 105 phWT4 cells and (F) 5 × 105 phWT147 cells were injected i.p. into 6 mice per group. Data is from one experiment. Transplant experiments demonstrated that the tumor lines from p53+∕−.Jα18−∕− mice grew in Jα18−∕− hosts, but were largely rejected in WT mice. Growth of these tumors in CD1d−∕− recipients was variable between tumors. The two tumor lines from p53+∕−.WT mice grew equivalently in all recipients.
- Figure S5. Comparative susceptibility of CD1d−∕− and Jα18−∕− mice to MCA-induced fibrosarcoma (JPG, 33.6 KB)
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Groups of 16–22 male mice were injected s.c. with 25 µg MCA. Following MCA-injection mice were monitored weekly for fibrosarcoma growth over a period of 250 days. Percentage survival of each group is plotted over time. Similar fibrosarcoma development in Jα18−∕− and CD1d−∕− mice was observed (incidence *, p = 1.00, Fishers exact test; survival curve, p = 0.2675 Log rank Mantel-Cox test).
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