Figure 1.

Long-Term Propagation of MRT Is Associated with an Increase in CSC Frequency

(A) Serial PDX propagation correlated with shorter time to tumor engraftment (mean of 26 days in early PDX passages in comparison with 16 days in late PDX passages. Results are presented as the mean of pooled data from early, intermediate, and late passages. p values were generated using the Mann-Whitney test, p < 0.001), indicating change in tumor behavior toward a more aggressive phenotype.

(B) Representative images of H&E staining of primary MRT-01 and late-passage PDX (P14). PDX tumors cells maintain the basic rhabdoid-like cellular morphology with some morphological differences, including the acquisition of spindle-like cells, vast areas of necrosis, less apoptotic bodies, and more mitoses (white arrows indicate mitoses). Scale bar, 100 μm (top) and 50 μm (bottom).

(C) Representative images of IHC of primary MRT-01, early-passage PDX (P2), intermediate-passage PDX (P7), and late-passage PDX (P14) in serial sections for cytokeratin (AE1/AE3), epithelial membrane antigen (EMA), neurofilament protein (NFP), and vimentin. Late-passage PDX showed loss of differentiation markers (top three panels) while primary tumor and PDX tissues strongly express vimentin (bottom panel). Specifically, vimentin staining was 70% in primary tumor as well as in P14 while staining of cytokeratin decreased from 10% to 5%, EMA decreased from 30% to 0, and NFP decreased from 50% to 5% in primary tumor in comparison with P14, accordingly. Scale bar, 200 μm.

(D) qRT-PCR analysis comparing the expression levels of E-cadherin and vimentin between primary tumor, early-passage PDX (P3), and late-passage PDX (P17). The expression of the epithelial marker, E-cadherin, is downregulated throughout serial propagation while the expression of vimentin remains permanent. For qRT-PCR analyses, the values for primary tumor cells were used to normalize (therefore = 1), and all other values were calculated with respect to them. Results are presented as the mean ± SEM of triplicates on three separated experiments. p values were generated using a two-tailed unpaired t test. ^∗p < 0.05.

(E) Representative flow cytometry analysis of PDX cells from early, intermediate and late MRT PDX (MRT-01) passages for the expression of several CSC markers including CD24, CD34, CD90, CD56, and ALDH1 antigens on single-cell suspensions. The results revealed that ALDH1 is a good candidate for MRT CSC marker, presenting a pattern of increased expression through PDX serial propagation. Results are presented as the mean of three separated experiments.

See also Figures S1 and Tables S1–S3.