Hypoxia and 3D Culture

This vignette from the R package canprot reproduces calculations of compositional oxidation state and hydration state that are described in a paper published in PeerJ (Dick, 2017).

Abbreviations

U937 (acute promonocytic leukemic cells), B104 (rat neuroblastoma cells), DU145 (prostate carcinoma cells), SK-N-BE(2)c; IMR-32; SH-SY5Y (neuroblastoma cells), H9C2 (rat heart myoblast), MCF-7 (breast cancer cells), THP-1 (macrophages), A431 (epithelial carcinoma cells), Hx48 (hypoxia 48 h), Hx72 (hypoxia 72 h), ReOx (hypoxia 48 h followed by reoxygenation for 24 h), -S (supernatant fraction), -P (pellet fraction), SPH (spheroids), HepG2/C3A (hepatocellular carcinoma cells), U87MG (glioblastoma), 786-O (renal clear cell carcinoma cells), HCT116; HT29 (colon cancer cells), SC (stem cells), SAL (salidroside).

Summary Table

This table compares the chemical compositions of groups of proteins that are relatively down- and up-expressed (n1 and n2, respectively) in cells grown in hypoxia or 3D culture compared to control conditions.

library(canprot)
data(canprot)
datasets <- pdat_hypoxia()
comptab <- lapply_canprot(datasets, function(dataset) {
  pdat <- get_pdat(dataset, "pdat_hypoxia")
  ZC_nH2O(pdat, plot.it=FALSE)
}, varlist="pdat_hypoxia")
library(xtable)
xsummary(comptab)
ZC nH2O
set reference (description) n1 n2 MD ES p-value MD ES p-value
a HXS+06 (U937) 37 24 -0.027 31 1e-02 0.029 62 1e-01
b BRA+10 (placental secretome) 41 22 -0.027 38 1e-01 0.011 54 6e-01
c DPL+10 (B104) 71 19 -0.021 37 9e-02 0.001 51 9e-01
d BMJ+11 (DU145) 87 28 0.014 61 8e-02 0.000 49 9e-01
e CBW+11 (SK-N-BE(2)c; IMR-32) 29 21 0.004 51 9e-01 0.001 50 1e+00
f LAR+12 (H9C2) 53 65 -0.007 51 8e-01 -0.066 35 6e-03
g MHG+12 (MCF-7 SPH P5) 409 337 -0.027 40 1e-06 -0.008 47 2e-01
h MHG+12 (MCF-7 SPH P2) 248 214 -0.025 42 2e-03 -0.003 48 5e-01
i MVC+12 (SPH perinecrotic) 48 52 -0.006 47 6e-01 -0.025 44 3e-01
j MVC+12 (SPH necrotic) 101 186 -0.023 38 9e-04 -0.029 43 6e-02
k FWH+13 (THP-1) 56 40 0.003 53 6e-01 0.011 54 5e-01
l RHD+13 (A431 Hx48) 178 77 0.012 53 4e-01 -0.017 46 4e-01
m RHD+13 (A431 Hx72) 69 54 -0.025 38 2e-02 -0.048 40 5e-02
n RHD+13 (A431 ReOx) 48 36 0.001 51 8e-01 0.003 56 4e-01
o VTMF13 (SH-SY5Y) 141 64 -0.021 39 1e-02 0.024 59 3e-02
p DYL+14 (A431 Hx48-S) 65 34 0.033 65 2e-02 -0.032 37 3e-02
q DYL+14 (A431 Hx72-S) 137 61 -0.006 49 8e-01 -0.012 45 2e-01
r DYL+14 (A431 ReOx-S) 56 49 0.037 67 4e-03 -0.050 33 2e-03
s DYL+14 (A431 Hx48-P) 74 44 -0.002 52 8e-01 -0.008 46 5e-01
t DYL+14 (A431 Hx72-P) 67 53 -0.013 46 4e-01 -0.015 43 2e-01
u DYL+14 (A431 ReOx-P) 41 31 0.021 64 4e-02 -0.035 37 6e-02
v RKP+14 (CRC-derived SPH) 113 154 0.012 55 2e-01 0.005 50 1e+00
w WRK+14 (HepG2/C3A SPH) 127 292 -0.032 39 4e-04 -0.029 43 2e-02
x BSA+15 (HeLa) 53 72 0.004 53 6e-01 -0.003 50 1e+00
y HWA+16 (U87MG and 786-O) 137 164 -0.001 49 8e-01 -0.026 42 2e-02
z LCS16 (HCT116 transcription) 129 141 -0.004 47 4e-01 0.024 57 4e-02
A LCS16 (HCT116 translation) 469 1024 -0.028 39 1e-11 -0.025 43 2e-05
B RSE+16 (adipose-derived SC) 66 50 0.031 67 2e-03 -0.011 45 3e-01
C XCJ+16 (cardiomyocytes CoCl2) 65 27 -0.025 41 2e-01 -0.012 46 6e-01
D XCJ+16 (cardiomyocytes SAL) 35 69 0.014 58 2e-01 -0.004 46 5e-01
E YLW+16 (HT29 SPH) 116 225 -0.053 29 2e-10 -0.018 45 2e-01

Data Sources

a. 2% O2 vs normoxic conditions. Source: Table 1 of Han et al. (2006). b. 1% vs 6% O2. Source: Tables 2 and 3 of Blankley et al. (2010). c. The comparisons here use expression ratios HYP/LSC (oxygen deprivation / low serum control) >1.2 or <0.83, calculated from the reported ratios. Source: extracted from Suppl. Table 2 of Datta et al. (2010), including proteins with p-value <0.05 and EF <1.4. d. Translationally regulated genes. Source: Suppl. Tables 1–4 of Beucken et al. (2011). e. 1% O2 for 72 h vs standard conditions. Source: Suppl. Table 1(a) of Cifani et al. (2011). f. Hypoxic vs control conditions for 16 h. Source: Suppl. Table S5 of Li et al. (2012). g. h. Tumourspheres (50 to 200 μm diameter) at passage 5 (P5) or 2 (P2) compared to adherent cells. Source: Sheets 2 and 3 in Table S1 of Morrison et al. (2012). i. j. Perinecrotic and necrotic regions compared to surface of multicell spheroids (~600 μm diameter). The comparisons here use expression ratios <0.77 or >1.3. Source: Suppl. Table 1C of McMahon et al. (2012). k. Incubation for several days under hypoxia (1% O2). Source: Suppl. Table 2A of Fuhrmann et al. (2013) (control virus cells). l. m. n. Source: extracted from Suppl. Table 1 of Ren et al. (2013), including proteins with iTRAQ ratios <0.83 or >1.2 and p-value <0.05. o. 5% O2 vs atmospheric levels of O2. The comparisons here include proteins with a normalized expression ratio of >1.2 or <0.83. Source: SI table of Villeneuve et al. (2013). p. q. r. s. t. u. The comparisons here include proteins with p <0.05. Source: Suppl. Table S1 of Dutta et al. (2014). v. Organotypic spheroids (~250 μm diameter) vs lysed CRC tissue. Source: extracted from Table S2 of Rajcevic et al. (2014), filtered as follows: at least two of three experiments have differences in spectral counts, absolute overall fold change is at least 1.5, and p-value is less than 0.05. w. SPH vs classical cell culture (2D growth). Standard concentrations of gases used for tissue culture (5% CO2, 95% air) were used in both cases. The comparisons here include proteins that have a log2 fold change of at least ±1. Source: P1_Data sheet in the SI of Wrzesinski et al. (2014). x. 1% vs 19% O2. Source: Table S1 of Bousquet et al. (2015). y. 1% O2 for 24 hr. The comparisons here include proteins with a fold change of <0.5 or >1 and proteins that were detected in only hypoxic or only normoxic conditions. Source: Table S1 of Ho et al. (2016). z. A. Microarray analysis of differential gene expression in the transcriptome (total rRNA) and translatome (polysomal / total RNA ratio) of cells grown in normal and hypoxic (1% O2) conditions. Source: data file supplied by Dr. Ming-Chih Lai (Lai, Chang & Sun (2016)). B. ASC from 3 donors cultured for 24 hr. in hypoxic (1% O2) vs normoxic (20% O2) conditions. Source: Tables 1 and 2 of Riis et al. (2016). C. D. Rat cardiomyocytes treated with CoCl2 (hypoxia mimetic) vs control or with SAL (anti-hypoxic) vs CoCl2. Source: SI Tables 1S and 2S of Xu et al. (2016). E. 800 μm spheroids vs 2D monolayers. Source: Tables S1a–b of Yue et al. (2016).

Mean Differences

The reoxygenation or anti-hypoxic, tumor spheroid, and adipose-derived stem cell datasets are highlighted in blue, red, and orange, respectively.

col <- rep("black", length(datasets))
col[grepl("ReOx", datasets)] <- "blue"
col[grepl("=SPH", datasets)] <- "red"
col[grepl("=ASC", datasets)] <- "orange"
diffplot(comptab, col=col)

References

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Blankley RT., Robinson NJ., Aplin JD., Crocker IP., Gaskell SJ., Whetton AD., Baker PN., Myers JE. 2010. A gel-free quantitative proteomics analysis of factors released from hypoxic-conditioned placentae. Reproductive Sciences 17:247–257. DOI: 10.1177/1933719109351320.

Bousquet PA., Sandvik JA., Arntzen MØ., Jeppesen Edin NF., Christoffersen S., Krengel U., Pettersen EO., Thiede B. 2015. Hypoxia strongly affects mitochondrial ribosomal proteins and translocases, as shown by quantitative proteomics of HeLa cells. International Journal of Proteomics 2015:678527. DOI: 10.1155/2015/678527.

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Xu Z-W., Chen X., Jin X-H., Meng X-Y., Zhou X., Fan F-X., Mao S-Y., Wang Y., Zhang W-C., Shan N-N., Li Y-M., Xu R-C. 2016. SILAC-based proteomic analysis reveals that salidroside antagonizes cobalt chloride-induced hypoxic effects by restoring the tricarboxylic acid cycle in cardiomyocytes. Journal of Proteomics 130:211–220. DOI: 10.1016/j.jprot.2015.09.028.

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