Figure 5.
S100A8/A9 expression is associated with enhanced therapeutic resistance. (A) AML cell lines (OCI-AML, n = 5; MOLM-13, n = 5) were incubated for 48 hours in the absence (−, circles) or presence (+, squares) of HS-5 CM and treated with 50 nM doxorubicin for the last 24 hours of culture. Viability was analyzed by annexin-V/7-AAD staining via flow cytometry, and specific cell death by doxorubicin was calculated. (B-C) Similarly, specific cell death by 50 nM doxorubicin of AML cell lines (OCI-AML, n = 3; MOLM-13, n = 2-5) cultured in the presence of HS-5 CM was analyzed in untreated (circles) and treated (squares) samples treated with ruxolitinib (B) or SSO (C). (D) The frequency of S100A8/A9hi OCI-AML (n = 8) and MOLM-13 (n = 11) cells was determined by flow cytometry after 48 hours of culture in the absence and presence of HS-5 CM and upon treatment with 50 nM doxorubicin for the last 24 hours, as indicated. (E-F) Frequency of S100A8/A9hi OCI-AML (n = 3-4) and MOLM-13 (n = 4) cells after culture for 48 hours in the presence of HS-5 CM and for the last 24 hours in the presence of 50 nM doxorubicin was determined by flow cytometry upon treatment with ruxolitinib (E) or SSO (F). (G) Free cytosolic calcium was semiquantified in AML cells (OCI-AML, n = 3; MOLM-13, n = 3) cultured for 48 hours in the absence (medium) or presence (CM) of HS-5 CM with the flow cytometric probe Fluo-8. (H) Specific cell death of AML cells (OCI-AML n = 3; MOLM-13 n = 2) by 50 nM doxorubicin was estimated via annexin-V/7-AAD staining in untreated samples and upon scavenging extracellular (EGTA) and free cytosolic (BAPTA) calcium. (I) Log2-transformed normalized counts of the S100A8 and S100A9 gene expression (from TCGA LAML data set) correlated with the log2-transformed normalized count of MCL1 by the Spearman test. (J) Cell death of S100A8/A9lo and of S100A8/A9hi AML cells (OCI-AML, n = 3; MOLM-13, n = 3) cultured for 24 hours in the presence of HS-5 CM was analyzed upon treatment with increasing concentrations of venetoclax by annexin-V/7-AAD staining in flow cytometry. (K) Frequency of S100A8/A9hi AML cells (OCI-AML, n = 2; MOLM-13, n = 2) cultured for 24 hours in the absence or presence of HS-5 CM was determined by flow cytometry after treatment with increasing concentrations of venetoclax . (L-M) Intracellular protein levels of Mcl-1 (L, OCI-AML, n = 4; MOLM-13, n = 4) and Bcl-2 (M, OCI-AML, n = 3; MOLM-13, n = 3) were measured by flow cytometry in S100A8/A9lo and S100A8/A9hi subsets of AML cells cultured for 48 hours in the presence of HS-5 CM. Data are expressed as the standard error of mean. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ns, not significant.

S100A8/A9 expression is associated with enhanced therapeutic resistance. (A) AML cell lines (OCI-AML, n = 5; MOLM-13, n = 5) were incubated for 48 hours in the absence (−, circles) or presence (+, squares) of HS-5 CM and treated with 50 nM doxorubicin for the last 24 hours of culture. Viability was analyzed by annexin-V/7-AAD staining via flow cytometry, and specific cell death by doxorubicin was calculated. (B-C) Similarly, specific cell death by 50 nM doxorubicin of AML cell lines (OCI-AML, n = 3; MOLM-13, n = 2-5) cultured in the presence of HS-5 CM was analyzed in untreated (circles) and treated (squares) samples treated with ruxolitinib (B) or SSO (C). (D) The frequency of S100A8/A9hi OCI-AML (n = 8) and MOLM-13 (n = 11) cells was determined by flow cytometry after 48 hours of culture in the absence and presence of HS-5 CM and upon treatment with 50 nM doxorubicin for the last 24 hours, as indicated. (E-F) Frequency of S100A8/A9hi OCI-AML (n = 3-4) and MOLM-13 (n = 4) cells after culture for 48 hours in the presence of HS-5 CM and for the last 24 hours in the presence of 50 nM doxorubicin was determined by flow cytometry upon treatment with ruxolitinib (E) or SSO (F). (G) Free cytosolic calcium was semiquantified in AML cells (OCI-AML, n = 3; MOLM-13, n = 3) cultured for 48 hours in the absence (medium) or presence (CM) of HS-5 CM with the flow cytometric probe Fluo-8. (H) Specific cell death of AML cells (OCI-AML n = 3; MOLM-13 n = 2) by 50 nM doxorubicin was estimated via annexin-V/7-AAD staining in untreated samples and upon scavenging extracellular (EGTA) and free cytosolic (BAPTA) calcium. (I) Log2-transformed normalized counts of the S100A8 and S100A9 gene expression (from TCGA LAML data set) correlated with the log2-transformed normalized count of MCL1 by the Spearman test. (J) Cell death of S100A8/A9lo and of S100A8/A9hi AML cells (OCI-AML, n = 3; MOLM-13, n = 3) cultured for 24 hours in the presence of HS-5 CM was analyzed upon treatment with increasing concentrations of venetoclax by annexin-V/7-AAD staining in flow cytometry. (K) Frequency of S100A8/A9hi AML cells (OCI-AML, n = 2; MOLM-13, n = 2) cultured for 24 hours in the absence or presence of HS-5 CM was determined by flow cytometry after treatment with increasing concentrations of venetoclax . (L-M) Intracellular protein levels of Mcl-1 (L, OCI-AML, n = 4; MOLM-13, n = 4) and Bcl-2 (M, OCI-AML, n = 3; MOLM-13, n = 3) were measured by flow cytometry in S100A8/A9lo and S100A8/A9hi subsets of AML cells cultured for 48 hours in the presence of HS-5 CM. Data are expressed as the standard error of mean. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ns, not significant.

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