ABSTRACT: Small RNA next generation sequencing of mice skin biopsies from acetone-treated healthy skins and DMBA/PMA-treated hyperplastic skins, papillomas and cutaneous Squamous Cell Carcinomas
Project description:FVB/N mice were subjected to 7,12-Dimethylbenz[a]anthracene (DMBA) two-hit multistage skin carcinogenesis protocol. Mice were topically treated with 200 nmol of DMBA in 0.2 mL acetone then twice weekly for six weeks with 5 nmol PMA (Phorbol 12-myristate 13-acetate). A second hit of DMBA was performed on the eighth week followed by the resumption of PMA treatment for 14 more weeks. Control mice were only topically treated with 0.2 mL acetone vehicle. Healthy skins (acetone-treated), hyperplastic skins, papillomas and tumors were harvested throughout the protocol and biopsies were frozen for RNA extraction.
Project description:FVB/N mice were subjected to 7,12-Dimethylbenz[a]anthracene (DMBA) two-hit multistage skin carcinogenesis protocol. Mice were topically treated with 200 nmol of DMBA in 0.2 mL acetone then twice weekly for six weeks with 5 nmol PMA (Phorbol 12-myristate 13-acetate). A second hit of DMBA was performed on the eighth week followed by the resumption of PMA treatment for 14 more weeks. Control mice were only topically treated with 0.2 mL acetone vehicle. Healthy skins (acetone-treated), hyperplastic skins, papillomas and tumors were harvested throughout the protocol and biopsies were frozen for RNA extraction. Small RNA libraries were generated from total RNAs of control skins, hyperplastic skins, papillomas and cSCCs biopsies (n=5 in each group) corresponding to a total of 20 samples, and sequenced on the Applied Biosystems SOLiD System.
Project description:Female FVB/N and Tg.Ac (hemizygotes) mice 3-5 weeks of age were purchased from Harlan UK and Taconic UK, respectively. The mice were contained in an isolator and maintained at 19C with 50% relative humidity and a 12-hour light-dark cycle. Following a 1 week acclimatisation period the dorsal skin of the mice was shaved (approximately 8cm2 area) 2 days prior to commencing treatment. FVB/N mice (5 per group) were treated with dimethylbenz[a]anthracene (DMBA) (10ug/200ul in acetone) or vehicle control one week before promotion with TPA. TPA (2.5ug/200ul in acetone) was applied to the back of both FVB/N and Tg.Ac mice (5 per group) twice a week for up to 14 weeks; control mice received acetone. Mice were shaved and examined weekly for the presence of papillomas. At each time point (FVB/N mice 2, 4, 6 and 14 weeks of TPA promotion and Tg.Ac mice 4 days, 1, 2 and 9 weeks of TPA promotion) the mice were killed by cervical dislocation and the dorsal skin removed, a representative portion was fixed in 10% neutral buffered formalin and retained for histological analysis; the remainder was snap frozen in liquid nitrogen.
Project description:RNA was prepared from papilomas of 2-3 mice (2 for WT and 3 for Knockout) and this was pooled to perform the array. Each pooled sample was run in triplicates. The mice were treated once with DMBA, followed by TPA application for 30 wks. These mice are mixed background of FVB and C57bl6 and the age when they got the papillomas was about 9 months. Keywords: Genetic modification
Project description:Germline polymorphisms influence gene expression networks in normal mammalian tissues. Analysis of this genetic architecture can identify single genes and whole pathways that influence to complex traits including inflammation and cancer susceptibility. Changes in the genetic architecture during the development of benign and malignant tumours have not been investigated. Here, we document major changes in germline control of gene expression during skin tumour development as a consequence of cell selection, somatic genetic events, and changes in tumour microenvironment. Immune response genes such as Interleukin 18 and Granzyme E are under germline control in tumours but not in normal skin. Gene expression networks linked to tumour susceptibility and hair follicle stem cell markers in normal skin undergo significant reorganization during tumour progression. Our data highlight opposing roles of Interleukin-1 signaling networks in tumour susceptibility and tumour progression and have implications for the development of chemopreventive strategies to reduce cancer incidence. Skin tumors were induced on dorsal back skin from a Mus spretus / Mus musculus backcross ([SPRET/Ei X FVB/N] X FVB/N) mice by treatment of dorsal back skin with dimethyl benzanthracene (DMBA) and tetradecanoyl-phorbol acetate (TPA). This treatment induced multiple benign papillomas as well as malignant squamous cell carcinomas (SCC) and spindle cell carcinomas. Gene expression analysis was performed on mRNA extracted from 68 papillomas: two papillomas from each of 31 FVBBX mice and a single papilloma from six additional FVBBX mice. Papillomas were harvested when mice were sacrificed due to presence of a carcinoma or termination of the experiment.
Project description:We used microarrays to assess the global gene expression profiles of cancer stem cells which were isolated from cutaneous squamous cell carcinomas which developed when WT, TGF beta receptor II ko, FAK KO, and TGF beta receptor II/FAK double KO were subjected to continuous DMBA treatment. Squamous cell carcinoma stem cells were compared to epidermal progenitor cells (CD49fhighCD34low) and hair follicle bulge stem cells (CD49fhighCD34high).
Project description:We profiled the transcriptomes of sorted neutrophils from precancerous and tumors in a DMBA/PMA cutenous squamous cell carcinoma, as well as the healthy adjacent tissue.
Project description:We used microarrays to assess the global gene expression profiles of cancer stem cells which were isolated from cutaneous squamous cell carcinomas which developed when WT, TGF beta receptor II ko, FAK KO, and TGF beta receptor II/FAK double KO were subjected to continuous DMBA treatment. Squamous cell carcinoma stem cells were compared to epidermal progenitor cells (CD49fhighCD34low) and hair follicle bulge stem cells (CD49fhighCD34high). Squamous cell carcinoma was initiated by continuous DMBA treatment (complete carcinogenesis). Cancer cells were sorted based on high CD29 and CD49f expression, infected with a retrovirus expressing a triple modality reporter (luciferase, RFP, thymidine kinase) and grafted onto nude recipient mice. RFP positive lineage was separated into CD29 high, CD49f high and CD34 low; or CD29high, CD49f high and CD34high squamous cell carcinoma initiating cells.
Project description:Germline polymorphisms influence gene expression networks in normal mammalian tissues. Analysis of this genetic architecture can identify single genes and whole pathways that influence to complex traits including inflammation and cancer susceptibility. Changes in the genetic architecture during the development of benign and malignant tumours have not been investigated. Here, we document major changes in germline control of gene expression during skin tumour development as a consequence of cell selection, somatic genetic events, and changes in tumour microenvironment. Immune response genes such as Interleukin 18 and Granzyme E are under germline control in tumours but not in normal skin. Gene expression networks linked to tumour susceptibility and hair follicle stem cell markers in normal skin undergo significant reorganization during tumour progression. Our data highlight opposing roles of Interleukin-1 signaling networks in tumour susceptibility and tumour progression and have implications for the development of chemopreventive strategies to reduce cancer incidence. Skin tumors were induced on dorsal back skin from a Mus spretus / Mus musculus backcross ([SPRET/Ei X FVB/N] X FVB/N) mice by treatment of dorsal back skin with dimethyl benzanthracene (DMBA) and tetradecanoyl-phorbol acetate (TPA). This treatment induced multiple benign papillomas as well as malignant squamous cell carcinomas (SCC) and spindle cell carcinomas. 60 carcinomas were harvested from 55 mice; five mice provided two carcinomas each.
