A vascular model of Tsc1 deficiency accelerates renal tumor formation with accompanying hemangiosarcomas

Abstract

Tuberous sclerosis complex (TSC) is an autosomal disease caused by inactivating mutations in either of the tumor suppressor genes TSC1 or TSC2. TSC-associated tumor growth is present in multiple tissues and organs including brain, kidney, liver, heart, lungs, and skin. In the kidney, TSC angiomyolipomas have aberrant vascular structures with abnormal endothelial cells, suggesting a role for endothelial mTORC1 function. In the current report, a genetically engineered mouse model (GEMM) with a conditional knockout allele of Tsc1 with a Darpp32-Cre allele displayed accelerated formation of both kidney cystadenomas and paw hemangiosarcomas. All mutant mice developed hemangiosarcomas on multiple paws by 6 weeks of age. By 16 weeks of age, the average mutant hind paw was 4.0 mm in diameter, nearly double the size of control mice. Furthermore, the hemangiosarcomas and kidney cystadenomas were responsive to intraperitoneal rapamycin treatment. Immunoblotting and immunostaining for phospho-S6 (pS6) and phospho-CAD showed that the effect of rapamycin on tumor size was through inhibition of the mTOR signaling pathway. Finally, elevated VEGF mRNA levels were also observed in hemangiosarcoma specimens. Because paw hemangiosarcomas are easily detectable and scorable for size and growth, this novel mouse model enables accelerated in vivo drug testing for therapies of TSC-related tumors.

Implications: These findings provide a strong rationale for simultaneous use of this conditional knockout mouse as an in vivo genetic model while seeking new cancer therapies for TSC-related tumors.

Figure 1

Loss of TSC1 in renal and paw tissue results in tumor formation. A) Representative images showing Lac-Z staining in 8 week old Tsc1cc Darpp-32-Cre+ kidney (left) and paw (right) tissue confirms recombination, 4X Scale bar = 500uM. Multiple tumor lesions are seen to have the blue stain. B) Tsc1cc Darpp-32-Cre+ mice show reduced mean (±SEM) weight from weaning through age 100 days. C) Tsc1cc Darpp-32-Cre+ mice have markedly reduced survival in comparison to wild type littermates. D) Gross anatomical photos taken from 17 week old Tsc1cw Darpp-32-Cre+ (left) and Tsc1cc Darpp-32-Cre+ (right) mice at sacrifice. Top highlights tumor formation in kidneys while bottom highlights increased paw size due to tumor formation. * indicates P < 0.05, ** indicates P < 0.01

Figure 2

Tsc1^cc DARPP Cre mouse paw pathology. A-F) Hematoxylin and Eosin staining of paw lesions show extensive clusters of aberrant vascular cells and multiple hemangiosarcomas. A–C) Paw from 8-week-old mutant mouse, at 2× (A), 20× (B), and 60× (C) magnification. Black arrows indicate paw hemangiosarcoma tissue. D–F) Paw from 8 week old wild type mouse at 2× (D), 10× (E), and 20× (F). 2× scale bar = 1 mm, 10× scale bar = 200 um, 20× scale bar = 100 um, 60× scale bar = 33 um.

Figure 3

Tsc1^cc DARPP Cre mouse renal pathology. A–C) Hematoxylin and Eosin staining of kidney lesions show extensive clusters of aberrant vascular cells and a variety of cystadenomas through adulthood. A) Kidney from 8-week-old mutant mouse, 2× magnification. B) Kidney from 8-week-old mutant mouse, 10× magnification. C) Kidney from 17-week-old mutant mouse, 4× magnification. Black arrows indicate cystadenomas. D) Kidney from 8-week-old wild type mouse, 2× magnification. E) Kidney from 8-week-old wild type mouse, 10× magnification. F) Kidney from 17-week-old wild type mouse, 4× magnification. 2× scale bar = 1 mm, 4× scale bar = 500um, 10× scale bar = 200 um.

Figure 4

Progressive growth of paw hemangiosarcomas and response to rapamycin. A) Paw hemangiosarcomas grow progressively as seen by the mean (±SEM) paw diameter of largest hemangiosarcoma across all mice. B) Effect of rapamycin treatment on mean (±SEM) paw tumor diameter in Tsc1^cc DARPP Cre mice. C) Immunoblotting of total S6-240, phospho-S6-S240, phospho-CAD, total CAD, and Tubulin expression in both paws and kidneys from wild type and Tsc1^cc DARPP Cre vehicle and rapamycin treated mice. D) Normalized quantification of expression of phospho-S6-240 relative to total S6-240 (top) and phospho-CAD relative to total CAD (bottom) in both paws and kidneys from wild type and Tsc1^cc DARPP Cre vehicle and rapamycin treated mice. n= 5 for phospho and total-S6-240 experimental groups, n=4 for each phospho and total-CAD experimental groups. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

Figure 5

Histological analysis of kidney cyst diameter and load in Tsc1^cc DARPP Cre mice. A) Mean cyst diameter in mm⁻³ measured in vehicle treated mice at 14 weeks, vehicle treated mice at 17 weeks, and rapamycin treated mice at 17 weeks. B) Mean cyst count measured in vehicle treated mice at 14 weeks, vehicle treated mice at 17 weeks, and rapamycin treated mice at 17 weeks. C) 4X magnification of 17-week old mutant vehicle treated mouse kidney showing extensive cyst formation with arrows highlighting the largest of the representative cysts. D) 4× magnification of 17-week old mutant rapamycin treated mouse kidney showing significantly reduced size of cysts, as highlighted by arrows. 4× scale bar = 500um. * indicates P < 0.05, *** indicates P < 0.001

Figure 6

Paw hemangiosarcomas and kidney cystadenomas response to rapamycin treatment. Immunostaining of phospho-S6-S240 expression (brown) in (A) kidneys and (B) paws after vehicle or rapamycin treatment, 4× scale bar = 500um, 20× scale bar = 100 um.

Figure 7

VEGF mRNA levels are elevated in Tsc1^cc DARPP Cre mouse paws and kidneys. A) Relative expression of VEGF-A mRNA transcripts harvested from 17-week old control and mutant paws and kidneys. B) Relative expression of VEGF-C mRNA transcripts harvested from 17-week old control and mutant paws and kidneys. C) Relative expression of VEGF-D mRNA transcripts harvested from 17-week old control and mutant paws and kidneys. All measurements are standardized to GAPDH. * indicates P < 0.05, *** indicates P < 0.001