Supplemental Material Modeling Selective Elimination of Quiescent Cancer Cells from Bone Marrow   Stephen P. Cavnara,b, Andrew D. Rickelmannc,d, Kaille F. Meguiarc,d, Annie Xiaoc,d, Joseph Doschc, Brendan M. Leunga,b, Sasha Cai Lesher-Pereza,b, Shashank Chittac,d, Kathryn E. Lukerc,e, Shuichi Takayamaa,b,e, Gary D. Lukera,c,d,f,§ Supplemental Figures S1-S4

Figure S1 Figure S1. Response of T47D and stromal cells to cytotoxic drugs. Dose-response cytotoxicity curves for T47D cells treated for eight days with cisplatin, doxorubicin, or paclitaxel in bone marrow spheroids. Red curves designate CBRed HS5 signal, and green curves designate the CBGreen cancer cell signal, respectively. After background subtraction, we normalized photon flux values for both CBGreen and CBRed luciferases in cancer and stromal cells, respectively, to their respective untreated control. We calculated selective toxicity for cancer cells based on the log-2-scale ratio of CBRed to CBGreen signal adjusted to center around the untreated control (black curve). Positive values for selectivity (black curve) that lie above the horizontal dotted line denote selective loss of bioluminescence from cancer cells. We omit selectivity data points for conditions where CBRed and/or CBGreen signal is below 1% of untreated control since instrument noise generates most of the signal. Figure S1. Response of T47D and stromal cells to cytotoxic drugs. Dose-response cytotoxicity curves for T47D cells treated for eight days with cisplatin, doxorubicin, or paclitaxel in bone marrow spheroids. Red curves designate CBRed HS5 signal, and green curves designate the CBGreen cancer cell signal, respectively. After background subtraction, we normalized photon flux values for both CBGreen and CBRed luciferases in cancer and stromal cells, respectively, to their respective untreated control. We calculated selective toxicity for cancer cells based on the log-2-scale ratio of CBRed to CBGreen signal adjusted to center around the untreated control (black curve). Positive values for selectivity (black curve) that lie above the horizontal dotted line denote selective loss of bioluminescence from cancer cells. We omit selectivity data points for conditions where CBRed and/or CBGreen signal is below 1% of untreated control since instrument noise generates most of the signal.

Figure S2 Figure S2. Representative cytotoxicity curves for selective inhibitors of Jak2 (AG-490), PI3-kinase (GDC-0941), AKT (MK-2206), and β2-adrenergic receptor (propranolol) with MDA-MB-231 cells in HS5 spheroids. Graph shows mean values + SEM (n = 8 per condition) for fold change in bioluminescence from MDA-MB-231 cells (green curves) and HS5 cells (red curves). We calculated selectivity of compounds for cancer versus stromal cells as described in Fig S1 (black curves) with positive values representing greater killing of cancer cells and negative selectivity values for greater killing of HS5 stromal cells. Figure S2. Representative cytotoxicity curves for selective inhibitors of Jak2 (AG-490), PI3-kinase (GDC-0941), AKT (MK-2206), and β2-adrenergic receptor (propranolol) with MDA-MB-231 cells in HS5 spheroids. Graph shows mean values + SEM (n = 8 per condition) for fold change in bioluminescence from MDA-MB-231 cells (green curves) and HS5 cells (red curves). We calculated selectivity of compounds for cancer versus stromal cells as described in Fig S1 (black curves) with positive values representing greater killing of cancer cells and negative selectivity values for greater killing of HS5 stromal cells.

Figure S3 Figure S3. Representative cytotoxicity curves for selective inhibitors of Jak2 (AG-490), PI3-kinase (GDC-0941), AKT (MK-2206), and β2-adrenergic receptor (propranolol) with T47D cells in bone marrow spheroids. Graph shows mean values + SEM (n = 8 per condition) for fold change in bioluminescence from MDA-MB-231 cells (green curves) and HS5 cells (red curves). We calculated selectivity of drugs for cancer versus stromal cells as described in Fig S1 (black curves) with positive values representing greater killing of cancer cells and negative selectivity values for greater killing of stromal cells. Figure S3. Representative cytotoxicity curves for selective inhibitors of Jak2 (AG-490), PI3-kinase (GDC-0941), AKT (MK-2206), and β2-adrenergic receptor (propranolol) with T47D cells in bone marrow spheroids. Graph shows mean values + SEM (n = 8 per condition) for fold change in bioluminescence from MDA-MB-231 cells (green curves) and HS5 cells (red curves). We calculated selectivity of drugs for cancer versus stromal cells as described in Fig S1 (black curves) with positive values representing greater killing of cancer cells and negative selectivity values for greater killing of stromal cells.

Figure S4 Figure S4. Combinatorial treatment with doxorubicin and Jak2 kinase inhibitor improves selective elimination of breast cancer cells. (A-B) Surface plots display cancer cell bioluminescence (CBGreen) normalized to control after 8 days of treatment (A) and again after 6 days post-treatment recovery (B) for MDA-MB-231 cells treated with the Jak2 inhibitor AG490 and doxorubicin. Color-scale reflects log-2-scale selectivity in panels A and B with positive values (red colors) depicting preferential loss of bioluminescence from cancer cells relative to HS5 stromal cells. Combinations of AG490 and doxorubicin produce the highest post-recovery within the circled region in panel C. Figure S4. Combinatorial treatment with doxorubicin and Jak2 kinase inhibitor improves selective elimination of breast cancer cells. (A-B) Surface plots display cancer cell bioluminescence (CBGreen) normalized to control after 8 days of treatment (A) and again after 6 days post-treatment recovery (B) for MDA-MB-231 cells treated with the Jak2 inhibitor AG490 and doxorubicin. Color-scale reflects log-2-scale selectivity in panels A and B with positive values (red colors) depicting preferential loss of bioluminescence from cancer cells relative to HS5 stromal cells. Combinations of AG490 and doxorubicin produce the highest post-recovery within the circled region in panel B.