![Figure 7. MyD88 deficiency impairs growth of v-rasHa–induced squamous tumors through an IL-1–independent signal. (A and B) Representative H&E micrograph of WT and MyD88−/− orthotopic grafts. WT or MyD88−/− primary v-rasHa–transduced keratinocytes combined with MyD88 WT dermal fibroblasts were grafted onto nude mice (A), and mean tumor volume was calculated as described in Materials and methods (B). Data shown are representative of two independent experiments and are reported as mean ± SEM. *, P < 0.05 between the WT and MyD88−/− group. Each group contains eight to nine mice. Only mice bearing tumors were included in this figure. (C) Tumors from A were stained for markers associated with tumor growth. Apoptotic cells were identified using the ApopTag kit, which stains nuclei containing nicked DNA. Positive nuclei were counted in five to seven randomly selected regions. (D) BrdU-labeled nuclei were detected in stained sections of tumors from mice injected with BrdU 1 h before sacrifice and similarly counted. (C and D) Data are reported as mean ± SEM. NS, difference not statistically significant between the WT and MyD88−/− groups. (E) Immunostaining for CD31 antigen outlining blood vessels within tumors originating from WT or MyD88−/− v-rasHa–transduced keratinocytes. (F) Microvessel density was determined based on the number of CD31+ cells in at least five fields per tumor originating from MyD88 WT or MyD88−/− v-rasHa–transduced keratinocytes. Data are reported as mean ± SEM. *, P < 0.05 between the WT and MyD88−/− groups. (G) Representative photographs of orthotopic grafts at the interscapular site (top) and midback site (bottom). 4 million RAS-keratinocytes were mixed with 5 million SENCAR mouse primary dermal fibroblasts before grafting. WT RAS-keratinocytes from C57BL/6J mice (IL-1R colony) underperform compared with WT RAS-keratinocytes from C57BL/6NCr mice (MyD88 colony) for both graft sites. In addition, the percentage of success of RAS-keratinocyte grafts from C57BL/6J mice at the midback graft site was very poor. WT and MyD88−/− and IL-1R−/− keratinocytes were derived from littermate pups. Grafting data presented in B, H, and J were performed at the midback site. Bars: (A and E) 50 µm; (G) 10 mm. (H) IL-1R WT or IL-1R−/− primary v-rasHa–transduced keratinocytes combined with SENCAR WT dermal fibroblasts were grafted onto nude mice, and mean tumor volume was calculated as described in Materials and methods. Each group contains three to five mice. Data shown are representative of two independent experiments and are reported as mean ± SEM. (I) BrdU-labeled nuclei and immunostaining for CD31 were analyzed as described for D and F. (J) v-rasHa–transduced keratinocytes (PBS or IL-1ra [Anakinra] treated) combined with SENCAR MyD88 WT dermal fibroblasts were grafted onto nude mice, and mean tumor volume was calculated as described in Materials and methods. PBS or Anakinra was administered daily by intraperitoneal injection starting the day after the surgical procedure for the duration of the experiment. Each group contains four to five mice. Data shown are representative of two independent experiments and are reported as mean ± SEM.](https://cdn.rupress.org/rup/content_public/journal/jem/209/9/10.1084_jem.20101355/4/jem_20101355_fig7.jpeg?Expires=1767717038&Signature=nuOa~cUKxIZA0nI-bhslmL86MX96vLRkySv31nQJmqsRxMqyY7S-EiBVflPYnwgAcffFzdZYWkbkEVgPqFHVK10VmQvVJ3Xg6e0HPx69MUi0vfxSkwn4R~zXavh3adz2J2RQyNiCWGivdAUwQfb1gNny8pMWi1yPTSYuUtfZ7m-f8euHkpmOAPLRZOAWEj0KV-94Wcwf9Gqhn5d4otIPbW4bdfYH1WLBxpqIYLE45i3LUxAfyJwhJ3sZ1yT-Efvgd-9Pb4UI7uBfVRlGgOf~XiiyU4U2KaT~6MBTHBiLG2GWv73iJW76cwFNTrl925uvHjH0TtR03vDtGuJgkEEHdA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
MyD88 deficiency impairs growth of v-rasHa–induced squamous tumors through an IL-1–independent signal. (A and B) Representative H&E micrograph of WT and MyD88−/− orthotopic grafts. WT or MyD88−/− primary v-rasHa–transduced keratinocytes combined with MyD88 WT dermal fibroblasts were grafted onto nude mice (A), and mean tumor volume was calculated as described in Materials and methods (B). Data shown are representative of two independent experiments and are reported as mean ± SEM. *, P < 0.05 between the WT and MyD88−/− group. Each group contains eight to nine mice. Only mice bearing tumors were included in this figure. (C) Tumors from A were stained for markers associated with tumor growth. Apoptotic cells were identified using the ApopTag kit, which stains nuclei containing nicked DNA. Positive nuclei were counted in five to seven randomly selected regions. (D) BrdU-labeled nuclei were detected in stained sections of tumors from mice injected with BrdU 1 h before sacrifice and similarly counted. (C and D) Data are reported as mean ± SEM. NS, difference not statistically significant between the WT and MyD88−/− groups. (E) Immunostaining for CD31 antigen outlining blood vessels within tumors originating from WT or MyD88−/− v-rasHa–transduced keratinocytes. (F) Microvessel density was determined based on the number of CD31+ cells in at least five fields per tumor originating from MyD88 WT or MyD88−/− v-rasHa–transduced keratinocytes. Data are reported as mean ± SEM. *, P < 0.05 between the WT and MyD88−/− groups. (G) Representative photographs of orthotopic grafts at the interscapular site (top) and midback site (bottom). 4 million RAS-keratinocytes were mixed with 5 million SENCAR mouse primary dermal fibroblasts before grafting. WT RAS-keratinocytes from C57BL/6J mice (IL-1R colony) underperform compared with WT RAS-keratinocytes from C57BL/6NCr mice (MyD88 colony) for both graft sites. In addition, the percentage of success of RAS-keratinocyte grafts from C57BL/6J mice at the midback graft site was very poor. WT and MyD88−/− and IL-1R−/− keratinocytes were derived from littermate pups. Grafting data presented in B, H, and J were performed at the midback site. Bars: (A and E) 50 µm; (G) 10 mm. (H) IL-1R WT or IL-1R−/− primary v-rasHa–transduced keratinocytes combined with SENCAR WT dermal fibroblasts were grafted onto nude mice, and mean tumor volume was calculated as described in Materials and methods. Each group contains three to five mice. Data shown are representative of two independent experiments and are reported as mean ± SEM. (I) BrdU-labeled nuclei and immunostaining for CD31 were analyzed as described for D and F. (J) v-rasHa–transduced keratinocytes (PBS or IL-1ra [Anakinra] treated) combined with SENCAR MyD88 WT dermal fibroblasts were grafted onto nude mice, and mean tumor volume was calculated as described in Materials and methods. PBS or Anakinra was administered daily by intraperitoneal injection starting the day after the surgical procedure for the duration of the experiment. Each group contains four to five mice. Data shown are representative of two independent experiments and are reported as mean ± SEM.
