Article Figures & Data
Figures
- Figure 1.
BV-resistant ALCL and Hodgkin lymphoma in vitro cell models. Proliferation experiments were performed in duplicate wells and averaged over 2 separate experiments, and a repeated measures ANOVA model is applied to show that time, group, and their interactions are all very significant (A and B). Viable cells counts were performed by hemocytometer with methylene blue. A, L428 and L428-R were seeded at 100,000 cells per well and incubated with BV at 80 μg/mL. L428-R cells are able to proliferate at this concentration whereas L428 cannot (P < 0.0001). B, Karpas-299 and Karpas-R were seeded at 40,000 cells per well and incubated with BV at 30 ng/mL. Karpas-R cells are able to proliferate at this concentration whereas Karpas-299 cannot (P < 0.0003). MTS assays were performed in triplicate wells and averaged over 3 separate experiments, and cells were seeded in 96-well plates at 5,000 cells (L428) and 10,000 cells (Karpas-299) per well. C, a four-parameter log-logistic model was fitted to assess inhibitory effect of L428-P and L428-R, respectively. The estimated IC50 SEs are 27.46 (4.76) and 236.08 (22.15), respectively. D, a four-parameter log-logistic model was fitted to assess inhibitory effect of Karpas-P and Karpas-R, respectively. The estimated IC50 SEs are 0.0029 (0.021) and 19.45 (1.87), respectively.
- Figure 2.
BV-resistant ALCL and Hodgkin lymphoma in vitro cell models. A–D, flow cytometry showing surface CD30 expression in Karpas-P, Karpas-R, and Karpas-R CD30+ and Karpas-R CD30− cells after cell sorting. E, CD30 mRNA expression by qRT-PCR (qRT-PCR performed with triplicate wells and repeated twice). A two-sample t test showed that there are significant expression differences for comparisons Karpas-P versus Karpas-R (P value = 0.003) and Karpas-R CD30+ versus Karpas-R CD30− (P value = 0.002). F, MTS assay was performed with triplicate wells and averaged over 3 experiments. A four-parameter log-logistic model was fitted to assess inhibitory effect of Karpas-P, Karpas-R, Karpas-R CD30+, and Karpas-R CD30−, respectively. The estimated IC50 SEs are 0.0058 (0.0071), 14.18 (1.46), 10.82 (0.89), and 16.99 (2.06), respectively.
- Figure 3.
CD30 staining in tissue from ALCL patients resistant to BV. Please see the Materials and Methods section on CD30 IHC staining. Biopsy after BV was done at the time of disease relapse while off BV or disease progression while on BV. CD30 expression is retained in both scenarios.
- Figure 4.
MMAE resistance and intracellular accumulation. A, MTS assay performed on L428-P and L428-R cells using MMAE. L428-R is resistant to MMAE as compared with L428-P (MTS assay performed in triplicate wells and repeated three times). A four-parameter log-logistic model was fitted to assess inhibitory effect of L428-P and L428-R, respectively. The estimated IC50 SEs are 0.63 (0.07) and 24.66 (4.87), respectively. B, MTS assay performed on Karpas-P and Karpas-R cells using MMAE. Karpas-R is equally sensitive to MMAE as compared with Karpas-P (MTS assay performed in triplicate wells and repeated three times). A four-parameter log-logistic model was fitted to assess inhibitory effect of Karpas-P and Karpas-R, respectively. The estimated IC50 SEs are 0.28 (0.05) and 0.52 (0.05), respectively. C–E, intracellular MMAE concentration from 0 to 48 hours of exposure with BV (experiments done in duplicate wells and repeated twice). Data are expressed as mean curves with 95% confidence interval and a repeated measures ANOVA model applied to show that the time, group, and their interactions are significant. C, L428-P had much more intracellular MMAE as compared with L428-R (P < 0.0001). D, Karpas-R and -P had same intracellular concentration of MMAE when incubated with low concentration of BV. E, Karpas-P had greater intracellular concentration of MMAE as compared with Karpas-R when incubated with higher concentration of BV (P ≤ 0.0001).
- Figure 5.
MDR1/P-glycoprotein drug exporter expressed in L428 BV-resistant model. A, MDR1 mRNA expression in L428-P, L428-R, and L428-R off drug for 5 months. qRT-PCR for MDR1 mRNA expression performed in triplicate wells and repeated three times. A two-sample t test showed that there is significant expression level difference for comparisons L428-P versus L428-R (P value < 0.0001), L428-P versus L428-R off (P value < 0.001), and L428-R off versus L428-R (P value = 0.003). B, Western blot for P-glycoprotein (PgP, protein product of MDR1 mRNA) in L428-P, L428-R, and L428-R off drug for 5 months. L428-R had more MDR1 mRNA and PgP as compared with L428-R off drug or L428-P. C, MTS assays performed in triplicate wells and averaged over 3 separate experiments. Cells were seeded in 96-well plates at 5,000 cells per well. A four-parameter log-logistic model was fitted to assess inhibitory effect of L428-R with and without verapamil, respectively. The estimated IC50s (±SE) are 76 (±23) and 297 (±12), respectively.
Tables
- Table 1.
Comparison of parental and BV-resistant cell lines
L428-P L428-R Karpas-P Karpas-R IC50 to BV 27 ± 4.8 μg/mL 236 ± 22 μg/mL 29 ± 21 ng/mL 19 ± 1.9 μg/mL Relative resistance 1 8.7 1 655 Cells CD30+ 98% 97% 96% 59% Median intensity CD30 280 ± 10 265 ± 5 592 ± 51 78 ± 17
Supplementary Data
Files in this Data Supplement:
- Supplemental Figure 1 - Supplemental Figure 1. Karpas-R cultured in the absence of BV regains CD30-positivity.
- Supplemental Figure 2 - Supplemental Figure 2. Karpas-R cultured in the absence of BV have decreased IC50.
- Supplemental Figure 3 - Supplemental Figure 3. Rhodamine Efflux Assay.
- Supplemental Figure 4 - Supplemental Figure 4. Immunohistochemistry for PgP on HL patient biopsy.
Volume 14, Issue 6
