Pipeline detailing the workflow of maRQup

a, A schematic of the in-vivo experimental setup used to evaluate the efficacy of CAR-T cell immunotherapy experiments. NALM-6 leukemia cells expressing luciferase were injected via the tail vein of mice followed by the adoptive transfer of CAR-T cells (IV) and the evaluation of tumor growth using a Xenogen IVIS Lumina, generating the output raw images for the maRQup pipeline. b, Processing steps to quantify tumor radiance at each timepoint for all mice in an experiment. IVIS output images are grouped by experimental condition (columns) and across multiple timepoints (rows) as indicated. Each output image contains a Brightfield (top) and Luminescent (bottom) image. Individual mice are then separated from the group image, and all mice images are aligned via image processing. Tumor burdens in different anatomical regions are quantified, and the tumor burden over time is calculated. c, Modeling, parametrization, and analysis of tumor behavior. Different phases of the tumor response dynamics — growth, decay, and relapse — are identified for each mouse. Each phase is modeled using differential equations, allowing for the rates of tumor growth in each phase to be compared across mice and experimental conditions.

Impact of CAR-T cell constructs and doses on in-vivo dynamics.

a, Schematic showing the extracellular and intracellular components of T cells harboring the CD19.4-1BB (left, blue) and the CD19.CD28 (right, orange) CAR constructs. b, Overall tumor response curves for all mice treated with CD19.4-1BB CAR-T cells (top) and CD19.CD28 CAR-T cells (bottom). c, The percentages of mice treated with CD19.4-1BB (blue) or CD19.CD28 (orange) CAR-T cells that exhibit an initial tumor growth (left) vs. those that relapse (right). d, Tree diagrams showing tumor behavior in mice treated with increasing doses of CD19.4-1BB CAR-T cells. e, The percentages of mice demonstrating an initial growth (top, green) as compared to a decay (bottom, blue) of NALM6 leukemia cells presented as a function of the dose of CD19.4-1BB CAR-T cells (<1, 1, 2, or >2 million). f, The percentages of leukemia-bearing mice that exhibit a final phase of relapse (top, red) as compared to decay (bottom, blue) presented as a function of the dose of CD19.4-1BB CAR-T cells.

Parametrization of tumor response to CAR-T cell therapy.

a, Tumor dynamics for all 1,060 mice in the dataset. Model calculations are shown as curves for all mice. A representative example is highlighted, with the experimental data shown as points. The growth, decay, and relapse phases are labeled in green, blue, and red, respectively. b, Residuals in log10-scale are shown for each timepoint in each phase. The percent of modeled points within one order of magnitude in either the positive or negative direction of the experimental points (dotted grey lines; 10−1 to 101) are labeled for each phase. c, Diagram displaying the parameters used to model the tumor dynamics. d, Comparison of the growth rate of NALM6 leukemia in the growth phase (kGrowth) and in the relapse phase (kRelapse) in mice that exhibited tumor growth, decay, and relapse. e, Tumor growth rates can be divided into slow growing (gold) and fast growing populations (purple). The vertical dotted line at 0.85 days−1 represents the division of the two populations. f, Tree diagram for the slow growing (left) and fast growing tumor populations (right). g, (left) Percentage of mice harboring slow or fast growing tumor populations as a function of their last phase being growth (NR), decay (CR/PR), or relapse (PD). (right) The percentages of mice that exhibit tumor decay and then relapse under conditions where the tumors exhibit slow (gold) or fast (purple) initial growth. h, Kernel density estimate plots of the distribution of the growth rate of tumors in the growth phase (kGrowth) (left), the rate of tumor killing by CAR-T cells (k − kGrowth) (middle), and the growth rate of tumors in the relapse phase (kRelapse) (right). Mice were classified into two groups of Effector to Target Ratios (E:T) depending on if the mice were treated with more (red) or fewer/equal (blue) CAR-T cells than tumor cells. The division of the slow (gold) and fast (purple) growing tumor populations, as determined from e is shown. i, Violin plots showing the growth rate of tumors during the relapse phase (kRelapse) as stratified by the initial behavior of the tumor: initial decay (kGrowth = 0), slow initial growth (0 < kGrowth ≤ 0.85), or rapid initial growth (kGrowth > 0.85)). All rates have units of days−1.

Characterizing spatial differences in tumor progression dynamics.

a, Zeros of four principal components measured across row-averaged and column-averaged mouse image data were used to demarcate nine distinct spatial regions. b, Images (top) and quantified radiance dynamics (bottom) both globally (all) and per region are shown for a representative mouse. c, Characterization of tumor growth dynamics in all mice engrafted with NALM6 leukemia (left) and the percentages of mice where tumor dynamics in a given region diverge from the dynamics evaluated from the overall total body radiance (right). The ‘bmR’ region is defined as the region containing the bone marrow of the mouse’s right leg; the ‘bmL’ region is defined as the region containing the bone marrow of the mouse’s left leg. d, Scatter plots of mice with a given overall tumor behavior as compared to the classified tumor behavior within each specific region. Residuals from the identity line (dashed diagonal line) with magnitudes outside the 95% confidence intervals (grey bands) indicate a significant divergence of a region specific growth pattern as compared to the overall growth pattern. e, Correlation coefficients for all pairwise combinations of overall tumor growth and region specific tumor growth.