Fig 1.
Sitravatinib target expression and activation increases in TKI-resistant metastatic tumor and non-tumor cells.
(A) Schematic showing in vivo derivation of SuR and AxR cell lines from metastatic lesions following surgical removal of primary tumors. (B) Whole genome expression analysis showing tyrosine kinase and sitravatinib target genes in 4T1AxR, RENCASuR, 3T3SuR, and LM2-4SuR cells compared to corresponding P cells (n = 3). (C) Representative western blots of phosphorylated and total protein levels of Met, EphA2, EphA3, and Axl in 4T1, RENCA, 3T3, and LM2-4 cells for P or SuR/AxR variants after treatment with 4μM sitravatinib for 3 or 24 hours. (D,E) Densitometric analysis of protein levels. Both phosphorylated and total protein levels were normalized to α-tubulin. (D) Protein levels in SuR/AxR variants were compared to corresponding P cells (n = 1–5). (E) Phosphorylation levels after treatment with 4μM sitravatinib for 3 hours (n = 1–3) compared to vehicle (DMSO) treated cells. Red area in bar graphs represent instances where sitravatinib treatment effect on phosphorylation levels is enhanced in SuR or AxR cells compared to sitravatinib treatment effect in P cells. Red numbers represent the enhanced effect (FC; log2) in SuR or AxR cells compared to P cells after sitravatinib treatment. P, parental; SuR, sunitinib resistant; AxR, axitinib resistant; FC, fold-change; TKI, tyrosine kinase inhibitor; Tx, treatment. ND, not done; n = 1, No error bars shown, n = 2, gray error bars shown; n>2, black error bars shown. Mean ± standard deviation (SD). 3T3SuR mouse fibroblast cells were derived in vitro by concentration escalation as described in ref 28. See S1 Appendix for original uncropped images of western blots shown. * p<0.05, ** p<0.01, compared to P cells.
Fig 2.
Enhanced proliferation inhibition in TKI-resistant cell lines after sitravatinib treatment.
(A-F) MTS proliferation assay for (A,E) 4T1 P/AxR, (B) RENCA P/SuR, (C) 3T3 P/SuR, (D) LM2-4 P/SuR, (F) 4T1 P/SuR (n = 3–4) after 72 hours treatment with different concentrations of sitravatinib (A-D) or cabozantinib (E,F). Proliferation curves (left panel) were used to calculate IC50 values (right panel). Proliferation values were normalized to DMSO-treated vehicle controls. (G) Corresponding IC50 values from MTS proliferation assays for P or SuR/AxR cell variants following sitravatinib or cabozantinib treatment (see S2 Fig for additional MTS proliferation assays). Orange boxes indicate significantly increased sensitivity to sitravatinib and blue boxes indicate significantly decreased sensitivity to cabozantinib treatment in SuR/AxR cells compared to P controls. (H) Annexin V staining of 4T1, RENCA, and 3T3 P or SuR/AxR cell variants after 2μM sitravatinib treatment for 48 hours (n = 3) using flow cytometric analysis. Results are normalized to vehicle-treated cells. P, parental; SuR, sunitinib-resistant; AxR, axitinib-resistant; FC, fold-change; Sig, significance; Mean ± standard deviation (SD); ** p<0.01, *** p<0.001, **** p<0.0001 compared to parental cells.
Fig 3.
Primary tumor inhibition following sitravatinib treatment is enhanced in TKI-resistant tumor models.
(A-F) Mice received orthotopic (mammary fat pad or kidney) implantation of P or SuR/AxR tumor cells. Tumor models used included 4T1P and 4T1AxR (Balb/c mice; n = 4–8); 4T1P and 4T1SuR (Balb/c mice; n = 4–5); RENCAP and RENCASuR (Balb/c mice; n = 4–17). Sitravatinib was administered to mice bearing (A-B) 4T1P and 4T1AxR, (C-D) 4T1P and 4T1SuR, and (E-F) RENCAP and RENCASuR. Treatment effects were assessed for impact on primary tumor growth by tumor volume or BL measurements (left panel) and for impact on metastasis after surgical tumor removal by survival (right panel). Treatment stopped the day before surgery. (G) Analysis of orthotopic primary tumor weights (or tumor bearing kidney weight) after sitravatinib treatment (includes LM2-4 tumor model data from S3 Fig). Bar graphs represent tumor burden of sitravatinib-treated P or SuR/AxR tumor cell variants compared to treatment-naïve P control cells (grey lines). Red area on bar graphs represent instances where sitravatinib treatment effect on tumor volume is enhanced in SuR/AxR tumors cell variants compared to sitravatinib-treated P tumors. Red numbers represent the enhanced effect (FC; log2) of sitravatinib treatment in resistant cells compared to their corresponding P cells. Tx, treatment; P, parental; SuR, sunitinib-resistant; AxR, axitinib-resistant; Su, sunitinib; Ax, axitinib; BL, bioluminescence; TKI, tyrosine kinase inhibitor; Mean ± standard deviation (SD); * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 compared to vehicle-treated mice. Significance stars in 3G: grey, represents Sitravatinib-treated vs. vehicle-treated; black, represents sitravatinib-treated SuR/AxR tumors vs. sitravatinib-treated P tumors. # represents disease-free survival until experiment end.
Fig 4.
Perioperative sitravatinib treatment improves survival in TKI-resistant models of spontaneous metastatic disease.
(A-B) Mice received orthotopic implantation in kidney of P or SuR tumor cells for RENCAP and RENCASuR (Balb/c mice; n = 5–8) models. Sitravatinib was administered to mice bearing (A) RENCAP, and (B) RENCASuR tumors and stopped at the end of the experiment. Post-surgical survival is shown. (C) Combined analysis showing differences in pre-surgical primary tumor and post-surgical metastatic burden for 4T1P and RENCAP (black dots); and 4T1AxR and RENCASuR (red dots) treated with vehicle or sitravatinib (Balb/c mice; n = 4). Crossed lines represent the standard deviation of sitravatinib-treated P (black cross) and SuR/AxR (red cross) cell-implanted tumor model data derived from comparisons of primary tumor burden data (vertical line) and median survival data (horizontal line). (D) Corresponding values for percent differences in primary tumor burden (tumor or tumor-bearing kidney weight) and overall survival (days post-implantation) are shown. Significance was assessed by student’s t test comparing SuR/AxR vs. P cell-implanted animals treated with sitravatinib and normalized to corresponding vehicle-treated animals. Tx, treatment; DPI, days post implantation; P; parental; SuR, sunitinib-resistant; AxR, axitinib-resistant; Su, sunitinib; Ax, axitinib; TKI, tyrosine kinase inhibitor; g, grams; Sig, significance. Mean ± standard deviation (SD); * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 compared to vehicle-treated mice or P cell-implanted model. # represents disease-free survival until experiment end (indicated as ‘censured’ in Fig 4C).