To further confirm, TUNEL assay was performed with tissue samples from experimental untreated and treated groups (Figure 8A,B), and the ratio of total (blue + brown) pixel to positive (brown) pixel of the total tissue was scanned and determined (= 3) to be five-fold (= 0

To further confirm, TUNEL assay was performed with tissue samples from experimental untreated and treated groups (Figure 8A,B), and the ratio of total (blue + brown) pixel to positive (brown) pixel of the total tissue was scanned and determined (= 3) to be five-fold (= 0.000168), further confirming the downregulation of apoptosis upon tamoxifen induction. Open in a separate window Figure 7 Tamoxifen treatment induced downregulation of apoptosis in KPC: APC mice. selected for APCf/f CDX2-Cre-ERT2 after the second round of inbreeding. The final model of the disease was generated by the cross of the two parental colonies and viable APC f/f KRAS +/f CDX2-Cre-ERT2 (KPC: APC) were genotyped and characterized. The model animals were tamoxifen (TAM) induced to generate tumors. Micro-positron emission tomography (PET) scan was used to detect and measure tumor volume and standard uptake value (SUV). Hematoxylin and eosin (H&E) staining was performed to establish neoplasm and immunohistochemistry (IHC) Tinostamustine (EDO-S101) was performed to determine histological similarities with human FFPE biopsies. The MSI/microsatellite stable (MSS) status was determined. Finally, the tumors were extensively characterized at the molecular level to establish similarities with human CRC tumors. The model Tinostamustine (EDO-S101) KPC: APC animals are conditional mutants that developed colonic tumors upon induction with tamoxifen in a dose-dependent manner. The tumors were confirmed to be malignant within four weeks of induction by H&E staining and higher radioactive [18F] fluoro-2-deoxyglucose (FDG) uptake (SUV) in micro-PET scan. Furthermore, the tumors histologically Tinostamustine (EDO-S101) and molecularly resembled human colorectal carcinoma. Post tumor generation, the KPC: APC animals died of cachexia and rectal bleeding. Implications: This model is an excellent preclinical platform to molecularly characterize the KRAS mutated colorectal tumors and discern appropriate therapeutic strategies to improve disease management and overall survival. = 8) (Figure S1B) post tamoxifen dosage while the positive control group survived at an average of 220 ILF3 days (= 9). The study was terminated at 250 days. Single high dose of tamoxifen at 1 mg/20 g body weight would result in rapid initiation of tumors with survival of an average of 15 days post induction in the (KPC: APC) experimental group. At tamoxifen dosage of 100 g per 20 g body weight the animal had an average of 24C30 days of latency before the tumor/focal lesion could be detected by PET/CT measurements. The Tamoxifen induced KPC: APC animals showed rapid disease progression during the last 25C30 days of their life (Figure S1B). Animals died with typical symptoms of rectal bleeding, significant loss of body weight, cachexia, morbidity, and particularly prominent kyphosis. Open in a separate window Open in a separate window Figure 1 (A) Schematic representation of the strategy adopted for the development of the KRAS mutated CRC mice. Essentially CDX2 ERT2 Cre mice were intercrossed with mice carrying loxP-flanked adenomatous polyposis coli (APC) alleles homozygous (APC loxP/loxP, 580S) or the loxP-Stop-loxP. The final model mice with tamoxifen (TAM)-inducible KRAS G12D expression (KPC: APC) was derived by breeding a Cre+/?. APC f/f with KRAS +/-APC f/f mice to generate APC f/f KRAS +/f CDX2-Cre-ERT2. (B) Tinostamustine (EDO-S101) Western blot analysis of active KRAS pull down in untreated (1 and 2) and treated (3 and 4) KPC: APC mice (= 2) shows higher KRAS activation detected in KPC: APC mice treated with tamoxifen. The expression of active KRAS in tamoxifen induced tumors was determined by pull down assay (= 2) (Figure 1B) prior to further characterization. 2.2. Gross Anatomy upon Dissection Profound inflammation of the cecum, ascending and transverse colon was observed upon tamoxifen induction in the KPC: APC experimental model (Figure Tinostamustine (EDO-S101) 2C). Multiple small tumors were visible throughout the entire inflamed region of the colon (Figure 2D,E) when the colon was dissected longitudinally to expose the mucosal layer. Although the positive control (CDX2 CRE ERT2 and APCf/f) showed enlargement and inflammation of the large bowel it was to a much lesser extent than the experimental model (Figure 2B). The negative control harboring KRAS+/? and APCf/f with no CDX2 CRE ERT2 showed no inflammation (Figure 2A). Open in a separate window Figure 2 The gross anatomical.