Anti-cancer capability of Eklf(K74R) mice as analyzed by the experimental melanoma metastasis assay.

(A) Flow chart illustrating the strategy of the pulmonary tumor foci assay. Left panels, representative photographs of pulmonary metastatic foci on the lungs of WT and Eklf(K74R) male mice in the B6 background two weeks after intravenous injection of B16-F10 cells (105 cells/ mouse). Statistical comparison of the numbers of pulmonary foci is shown in the two histograms on the right. N=10 (male) and N=7 (female), **, p<0.01. Note that only the numbers of large pulmonary foci (>1mm diameter) were scored. N>6, **, p<0.01. (B) Pulmonary tumor foci assay of 24 month-old WT and Eklf(K74R) male mice. Statistical comparison is shown in the two histograms. N=10 (male), *, p<0.05. (C) Pulmonary tumor foci assay of male mice in the FVB background. Statistical comparison is shown in the histograph on the right. N=10, **, p<0.01. (D) Pulmonary tumor foci assay of Eklf(K74A) male mice. Statistical comparison of the 3 month-old WT and Eklf(K74A) mice numbers of pulmonary foci is shown in the two histograms. N=10 (male), **, p<0.01. (E) The Eklf(K74R) mice and WT mice were subcutaneously injected with Hepa1-6 cells to form tumors. The tumor volumes were measured once a week using the formula: length x weight x height x π/6. The curves of tumor growth started to show difference between the Eklf(K74R) and WT mice at 49 days after Hepa1-6 cell injection. N>6, *p < 0.05, **p < 0.01 and ***p < 0.001.

Transfer of cancer resistance of Eklf(K74R) mice to WT mice by bone marrow transplantation (BMT)

(A) Flow chart illustrating the experimental strategy. (B) FACS analysis of the efficiency of BMT with use of 10Gy γ-irradiation. The percentages of CD45.1/CD45.2 cells in the PB of the recipient male mice were analyzed by flow cytometry, with the representative FACS charts shown on the left and the statistical histobar diagram on the right. (C) Transfer of the anti-metastasis capability of 8 week-old Eklf(K74R) male mice to age-equivalent WT male mice by BMT with use of 10Gy γ-irradiation. Left panels, representative photographs of lungs with pulmonary metastatic foci in the recipient WT (CD45.1) mice after BMT from WT (CD45.2) or Eklf(K74R) (CD45.2) donor mice and challenged with B16-F10 cells. Statistical analysis of the numbers of pulmonary B16-F10 metastatic foci on the lungs is shown in the right histogram. n=10, *, p<0.05. (D) Transplantation of 8 week-old male WT (CD45.1) mice with BMMNC from age-equivalent WT (CD45.2) male mice or from Eklf(K74R) (CD45.2) male mice with use of the γ-irradiation dosage 2.5Gy or 5Gy. The histobar diagram comparing the percentages of CD45.1 and CD45.2 PB cells of the recipient WT mice after BMT is shown on the left. The statistical analysis of the average numbers of pulmonary foci on the lungs of recipient WT mice after BMT and injected with the B16-F10 cells is shown in the right histogram, N=6. **, p<0.01, ***, p<0.001.

Inhibition of tumor growth in WT mice by BMT from Eklf(K74R) mice

(A) A flow chart of the experiments. Luciferase-positive B16-F10 cells were injected into the tail vein of 8 week-old WT male mice (day 0). The mice were then transplanted with BMMNC from WT or Eklf(K74R) male mice on day 11 after the luciferase-positive B16-F10 cell injection. In vivo imaging system (IVIS) was used to follow the tumor growth in mice on day 0, 10, 17 and 24, respectively. (B) Representative images of bioluminescence reflecting the luciferase activity from melanoma cancer cells in mice. The color bar indicates the scale of the bioluminescence intensity. (C) Statistical analysis of the intensities of bioluminescence in the cancer-bearing mice (WT◊WT, purple, N=7; Eklf(K74R)◊WT, blue, N=8; Control (no BMT), red, N=3).

Decrease of Pd-1 and Pd-l1 expression in blood cells of Eklf(K74R) mice

(A) Levels of Pd-1 and Pd-l1 mRNAs in the PB of WT and Eklf(K74R) male mice at the ages of 3 months and 24 months, respectively, as analyzed by RT-qPCR. Note the relatively low levels of Pd-1 and Pd-l1 mRNAs in the Eklf(K74R) mice at both ages in comparison to the WT mice. (B) Upper panels, comparison of the mRNA levels of Pd-1 and Pd-l1 of CD3+ T cells and B220+ B cells isolated from the PB of 8 week-old WT and Eklf(K74R) male mice. N=5. *, p<0.05; **, p<0.01. Lower panels, comparison of the protein levels of PD-1 and PD-L1, as analyzed by flow cytometry, of CD3+ T cells and B220+ B cells from 8 week-old WT and Eklf(K74R) male mice. N=3. *, p<0.05; **, p<0.01. (C) Comparison of the levels of Pd-1, Pd-l1 and Eklf mRNAs, as analyzed by RT-qPCR, in CD3+ T cells, which were isolated from splenocytes, without or with RNAi knockdown of Eklf mRNA. Two oligos (oligo-1 and oligo-2) were used to knockdown Eklf mRNA by ∼60-70%, which resulted in the reduction of Pd-1 mRNA level by 30-60% and nearly complete depletion of Pd-l1 mRNA. Control, T cells transfected with GFP-plasmid. SC, T cells transfected with scrambled oligos. N>3. *, p<0.05; **, p<0.01; ***, p<0.001.

Proteomics analysis of mouse leukocytes

(A) Heatmap plots representing age-dependent differentially expressed proteins (DEPs) in the leukocytes from the WT male mice (left) and Eklf(K74R) male mice (right). Differential Test Threshold: expression fold change > 1.5 and p-value < 0.01. (B) and (C), pathway analysis of the age-dependent DEPs changed in the concordant (B) and reverse (C) directions, respectively, in the WT and Eklf(K74R) mice. NES, normalized enrichment score. (D) Heatmap plots representing strain-dependent DEPs in leukocytes of 3 month-old (left) and 24 month-old (right) mice. (E) Pathway analyses of the strain-dependent DEPs in elder WT leukocytes (enrichment indicated by blue color) and elder Eklf(K74R) leukocytes (enrichment indicated by red color). The aging- and cancer-associated pathways are presented in the left and right diagrams, respectively. NES, normalized enrichment score. (F) A model of the regulation of the mouse lifespan by different cellular pathways in the leukocytes. The inter-relationship of 11 aging-associated cellular pathways differentially expressed in the leukocytes of Eklf(K74R) male mice in comparison to the WT male mice, among themselves and with respect to the regulation of lifespan (for references, see text), are depicted here. The directions and lengths of the arrows indicate changes of the individual pathways, up-regulation (upward arrows) or repression (downward arrows), in the leukocytes during aging of the WT male mice (blue arrows) and Eklf(K74R) male mice (red arrows), respectively.

Higher in vitro cancer cell cytotoxicity of NK(K74R) cells than WT NK cells

B16-F10 and Hepa1-6 cells were labeled with Calcein-AM (BioLegend) and co-cultured with NK cells in different E:T ratio for 4hrs. Killing rates of B16-F10 and Hepa 1-6 cancer cells by NK cells were determined by the intensities of fluorescence in the medium in comparison to the controls of spontaneous release and maximal release.