Figure 3.
Figure 3. Follicular tolerance of Hras is maintained long-term in uninjured tissue. All epithelial nuclei are in green (K14H2BGFP), and recombined cells are in red (tdTomato). (A) Two-photon images of the epidermis (left) and hair follicles (right) of a K19CreER;HrasG12V/+;K14H2BGFP;tdTomato mouse 1 yr after activation reveal that the skin appears grossly normal and the mutant cells (in red) are still present yet contained to the HFSC niche. (B) Two-photon revisit images of the same K19CreER;HrasG12V/+ follicles immediately after dermal papilla ablation (day 0, labeled with an asterisk) and at the indicated time points up to 13 mo. Mutant cells (in red) remain present yet contained to the HFSC niche and the ablated follicles never grow unlike their unablated neighbor. (C) Tumor emergence graph for K19CreER;Hras+/+ (n = 0/9), K19CreER;HrasG12V/+ (n = 2/10, well differentiated cSCCs), and K19CreER;HrasG12V/+;TGFβfl/fl (n = 5/7, poorly differentiated cSCCs) mice monitored over the course of 1 yr after activation. (D) Hematoxylin and eosin images of K19CreER;Hras+/+ (top left) and K19CreER;HrasG12V/+ (bottom left) clinically normal back skin from 1-yr-old mice. On the right are representative images of a tumor developed in a K19CreER;HrasG12V/+ mouse 1 yr after activation. The staining reveals verrucous squamous proliferation with acanthosis, hyperkeratosis and mild atypia consistent with well-differentiated squamous cell carcinoma. (E) Photograph of a cSCC tumor from a K19CreER;HrasG12V/+;TGFβfl/fl mouse 4 mo after tamoxifen activation. (F) Hematoxylin and eosin images revealing islands of atypical keratinocytes within the dermis extending into subcutaneous fat with marked atypia and associated mixed inflammatory infiltrate consistent with moderately differentiated, necrotic invasive squamous cell carcinoma. (G) Two-photon images of the ear skin from the same mouse from E and F at day 0 and 4 mo after tamoxifen activation reveal normal follicular architecture despite the persistent presence of mutant cells (tdTomato+). Follicles are labeled A–H as landmarks. Scale bars in two-photon images represent 50 µm. Scale bars in histology images represent 200 µm.

Follicular tolerance of Hras is maintained long-term in uninjured tissue. All epithelial nuclei are in green (K14H2BGFP), and recombined cells are in red (tdTomato). (A) Two-photon images of the epidermis (left) and hair follicles (right) of a K19CreER;HrasG12V/+;K14H2BGFP;tdTomato mouse 1 yr after activation reveal that the skin appears grossly normal and the mutant cells (in red) are still present yet contained to the HFSC niche. (B) Two-photon revisit images of the same K19CreER;HrasG12V/+ follicles immediately after dermal papilla ablation (day 0, labeled with an asterisk) and at the indicated time points up to 13 mo. Mutant cells (in red) remain present yet contained to the HFSC niche and the ablated follicles never grow unlike their unablated neighbor. (C) Tumor emergence graph for K19CreER;Hras+/+ (n = 0/9), K19CreER;HrasG12V/+ (n = 2/10, well differentiated cSCCs), and K19CreER;HrasG12V/+;TGFβfl/fl (n = 5/7, poorly differentiated cSCCs) mice monitored over the course of 1 yr after activation. (D) Hematoxylin and eosin images of K19CreER;Hras+/+ (top left) and K19CreER;HrasG12V/+ (bottom left) clinically normal back skin from 1-yr-old mice. On the right are representative images of a tumor developed in a K19CreER;HrasG12V/+ mouse 1 yr after activation. The staining reveals verrucous squamous proliferation with acanthosis, hyperkeratosis and mild atypia consistent with well-differentiated squamous cell carcinoma. (E) Photograph of a cSCC tumor from a K19CreER;HrasG12V/+;TGFβfl/fl mouse 4 mo after tamoxifen activation. (F) Hematoxylin and eosin images revealing islands of atypical keratinocytes within the dermis extending into subcutaneous fat with marked atypia and associated mixed inflammatory infiltrate consistent with moderately differentiated, necrotic invasive squamous cell carcinoma. (G) Two-photon images of the ear skin from the same mouse from E and F at day 0 and 4 mo after tamoxifen activation reveal normal follicular architecture despite the persistent presence of mutant cells (tdTomato+). Follicles are labeled A–H as landmarks. Scale bars in two-photon images represent 50 µm. Scale bars in histology images represent 200 µm.

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