FIGURE 7 from Statistical Assessment of Drug Synergy from <i>In Vivo</i> Combination Studies Using Mouse Tumor Models

A unified view of in vitro and in vivo synergy. Drug efficacy measurement, study design, and CI calculation are illustrated for the typical fixed-ratio in vitro combination study (A–C) and 4-group in vivo combination study (D–F). A, In in vitro cell assays, drug efficacy is measured at a single timepoint (e.g., 72 hours, the red dashed line) for a series of drug concentrations, where higher concentrations exert stronger antiproliferative effect as quantified by confluence percentage. B, In an in vitro combination study with the fixed-ratio design, dose–response curves are inferred for drugs A, B, A+B, using efficacy measured at 72 hours. C, The CI is estimated for a range of drug concentrations where high concentration causes higher fraction of affected (FA) cells. Therefore, the CI is dose dependent. D, In in vivo studies, drug efficacy is measured at a fixed dose (growth curve pointed by the red arrow) for the study duration. E, In an in vivo combination study with the 4-group design, tumor growth curves were obtained for vehicle control, drugs A, B, and A+B, each with a fixed dose. CI can be calculated at a particular day (cf. Eq. 4). F, The CI is calculated for a range of days in the study duration. Therefore, the CI is time dependent. We note that exponential growth kinetics is assumed for both cell proliferation (A) and tumor growth (D) under drug treatment, and well before reaching maximal values constrained by nutrient and space. Previous studies have provided theoretical (equation 5 in ref. 33) and empirical justifications (31).</p

FIGURE 5 from Statistical Assessment of Drug Synergy from <i>In Vivo</i> Combination Studies Using Mouse Tumor Models

Assessing synergy with delayed treatment effect. A, A 4-group in vivo combination study of the CDK4/6 inhibitor palbociclib and the EGFR inhibitor cetuximab in a colon PDX model CR1197, delayed treatment effect was observed in the combo treatment group. There are 10, 8, 9, 10 mice in the vehicle, cetuximab, palbociclib, and the combo groups, respectively. B, No synergy under the Bliss independence model was reported by the CombPDX method. C and D, Strong synergistic effect under the Bliss independence model was detected by the invivoSyn method, as evidenced by both the CI and SS. E and F, Simulations were conducted to obtain empirical statistical power to detect Bliss synergy with respect to the number of mice n in a group, assuming equal number of mice in all four groups, but with delayed treatment effect in the A+B combo group such that tumors exhibit reduced GR after day 10 at which drug effect takes place. TV is measured twice a week for 3 weeks (on days 0, 4, 7, 10, 14, 18, 21) assuming that tumor DT is 7 days, and the TV cutoff is set to 3,000 mm3. The simulation results show that sufficient power (>0.8) is only achieved with long study durations [i.e., observation days or Tobs = 40 and 50 days]. In C and D, the histogram, overlaid by a red fitted density curve, shows the distribution of 1,000 bootstrap values for CI or SS; the red dashed vertical line indicates additive effect (1 for CI and 0 for SS); the black triangle marks the calculated value for CI or SS; the blue horizontal line indicates the 95% confidence interval. invivoSyn is the name of our method as well as the software package implementing it.</p

FIGURE 3 from Statistical Assessment of Drug Synergy from <i>In Vivo</i> Combination Studies Using Mouse Tumor Models

Determining drug combination effects directly from in vivo studies. Combination effects were determined for an anti-mouse PD-1 antibody and five therapeutic agents that deplete tumor-infiltrating leukocytes in four syngeneic models (cancer types: CT26 colon, EMT6 breast, Hepa16 liver, MC38 colon). aCD25: antibody for CD25+ cells enriched for Treg; aCD4: antibody for CD4+ T cells; aCD8: antibody for CD8+ T cells; aNK: antibody for removing NK cells; Liposomes: clodronate liposomes for depleting TAM. CI values (blue dots) and 95% confidence intervals (black lines) were estimated under the Bliss independence model by the invivoSyn method, the red dashed line (CI = 1) indicates additive effect, CI > 1 is antagonistic effect, CI < 1 is synergistic effect.</p

FIGURE 4 from Statistical Assessment of Drug Synergy from <i>In Vivo</i> Combination Studies Using Mouse Tumor Models

invivoSyn detects synergy with high statistical power and low FDR under regular tumor growth patterns. A and B, Simulations were conducted to obtain empirical statistical power to detect Bliss synergy with respect to the number of mice n in a group, assuming equal number of mice in all four groups, under regular tumor growth where tumors in a group either grow or shrink with variations in GR. TV is measured twice a week for 3 weeks (on days 0, 4, 7, 10, 14, 18, 21) assuming that tumor DT is 7 days, and the TV cutoff is set to 3,000 mm3. C, Simulation results at four DT values (DT = 3, 5, 7, 10 days) with all other simulation parameters same as in A, showing that power increases with respect to DT for a given n value for both weak and strong synergies, while for nil synergy, the power is always low. D, Simulation results under five tumor volume cutoffs (500, 1,000, 1,500, 2,000, 2,500 mm3) for mouse euthanasia with DT = 5 days, showing that power is not strongly affected by TV cutoff.</p

FIGURE 1 from Statistical Assessment of Drug Synergy from <i>In Vivo</i> Combination Studies Using Mouse Tumor Models

The quantification of in vivo synergy based on an unbiased metric of drug effect. A, Definition of eGR, an unbiased metric of in vivo drug effect. Upper graphs shows two synthetic curves, for illustrative purposes, depicting tumor growth and shrinkage measured by TV. Bottom graphs show the same tumor growth curves in natural log scale, and illustrate the calculation of eGR, which is defined as , where AUC is the size of the colored area and d is the study duration in days (see Materials and Methods for details). B, The 4-group design is the most common in vivo combination study, which has four treatment groups for the vehicle control, drug A, drug B, and drug A+B, with fixed doses for the two drugs. A group usually has multiple mice that vary in tumor growth curves and number of TV datapoints. C, The average tumor growth curves of the four groups. The relative survival is calculated for drugs A, B, A+B based on TV at a particular day, then the CI and SS are estimated under several models, only Bliss independence model is shown. D, Bootstrap confidence internals and P values are calculated for both CI and SS. The histogram, overlaid by a red fitted density curve, shows the distribution of 1,000 bootstrap values for CI or SS; the red dashed vertical line indicates additive effect (1 for CI and 0 for SS); the black triangle marks the calculated value for CI or SS; the blue horizontal line indicates the 95% confidence interval. invivoSyn is the name of our method as well as the software package implementing it.</p

FIGURE 6 from Statistical Assessment of Drug Synergy from <i>In Vivo</i> Combination Studies Using Mouse Tumor Models

Assessing synergy with tumor regrowth. Simulations were conducted to obtain empirical statistical power to detect Bliss synergy with respect to the number of mice n in a group, assuming equal number of mice in all four groups but with tumor regrowth. TV is measured twice a week for 3 weeks (on days 0, 4, 7, 10, 14, 18, 21) assuming that tumor DT is 7 days, and the TV cutoff is set to 3,000 mm3. No synergy is imposed. A and B, Tumors regrow under combo treatment due to intrinsic resistance where 10% of tumor cells are intrinsically resistant to treatments A and A+B. C and D, Tumors regrow under combo treatment due to induced resistance where 10% of tumor cells resume growth with initial GR at day 10 under treatments A and A+B. In both scenarios, empirical power exhibits dependence on both mouse number n and observation time Tobs. When Tobs is sufficiently long (40–60 days), invivoSyn shows low empirical power, therefore low FDR, for synergy detection.</p

FIGURE 2 from Statistical Assessment of Drug Synergy from <i>In Vivo</i> Combination Studies Using Mouse Tumor Models

Validating in vitro drug synergy in in vivo combination studies. The TOP1 inhibitor irinotecan and the CHEK1 inhibitor rabusertib showed strong synergy in three colon cell lines, and were further tested in corresponding cell line–derived xenografts (4). The in vivo combination effects were reanalyzed by the CombPDX and invivoSyn methods for the three xenografts: SW837(A–D); SNU-81 (E–H); LS-1034 (I–L). For each xenograft, four graphs from left to right sequentially show tumor growth curves (A, E, I); local CI and gCI values with 95% confidence intervals and P values under the Bliss independence model by the CombPDX method (B, F, J); density distribution of the Bliss CI values based on 1,000 bootstrap resamples, along with the actual CI value (black triangle) as well as its 95% confidence interval (blue line) and P value, all by our invivoSyn method (C, G, K), where a red dashed vertical line indicates additive effect (1 for CI and 0 for SS); similar information for Bliss SSs by the invivoSyn method (D, H, L). Results in B, F, and J were drawn using output from the CombPDX website. Blue dots indicate local CI values, vertical lines show the 95% confidence interval. Some local CI values are erroneously not in the corresponding 95% confidence intervals.</p

Table S2 from Mouse Stromal Cells Confound Proteomic Characterization and Quantification of Xenograft Models

Gene set enrichment analysis results using the top 1000 DEPs between the original and de-moused tumors for each of five PDX models.</p

Table S1 from Mouse Stromal Cells Confound Proteomic Characterization and Quantification of Xenograft Models

Protein quantification results in cell line mixtures and PDX tumors.</p

Supplementary Figure 4 from Tumor Purity in Preclinical Mouse Tumor Models

The distribution of tumor purity in human cells of 2115 PDX models within passage 10.</p