Microenvironmentally-driven Plasticity of CD44 isoform expression determines Engraftment and Stem-like Phenotype in CRC cell lines

Theranostic biomarkers for putative cancer stem-like cells (CSC) in colorectal cancer (CRC) are of particular interest in translational research to develop patient-individualized treatment strategies. Surface proteins still under debate are CD44 and CD133. The structural and functional diversity of these antigens, as well as their plasticity, has only just begun to be understood. Our study aimed to gain novel insight into the plasticity of CD133/CD44, thereby proving the hypothesis of marker-associated tumorigenic and non-tumorigenic phenotypes to be environmentally driven. Methods: CD133/CD44 profiles of 20 CRC cell lines were monitored; three models with distinct surface patterns in vitro were systematically examined. CD133/CD44 subpopulations were isolated by FACS and analyzed upon in vitro growth and/or in limiting dilution engraftment studies. The experimental setup included biomarker analyses on the protein (flow cytometry, Western blotting, immunofluorescence) and mRNA levels (RT-/qPCR) as well as CD44 gene sequencing. Results: In general, we found that (i) the in vitro CD133/CD44 pattern never determined engraftment and (ii) the CD133/CD44 population distributions harmonized under in vivo conditions. The LS1034 cell line appeared as a unique model due to its de novo in vivo presentation of CD44. CD44v8-10 was identified as main transcript, which was stronger expressed in primary human CRC than in normal colon tissues. Biomarker pattern of LS1034 cells in vivo reflected secondary engraftment: the tumorigenic potential was highest in CD133+/CD44+, intermediate in CD133+/CD44- and entirely lost in CD133-/CD44- subfractions. Both CD44+ and CD44- LS1034 cells gave rise to tumorigenic and non-tumorigenic progeny and were convertible - but only as long as they expressed CD133 in vivo. The highly tumorigenic CD133+/CD44(v8-10)+ LS1034 cells were localized in well-oxygenated perivascular but not hypoxic regions. From a multitude of putative modulators, only the direct interaction with stromal fibroblasts triggered an essential, in vivo-like enhancement of CD44v8-10 presentation in vitro. Conclusion: Environmental conditions modulate CD133/CD44 phenotypes and tumorigenic potential of CRC subpopulations. The identification of fibroblasts as drivers of cancer-specific CD44 expression profile and plasticity sheds light on the limitation of per se dynamic surface antigens as biomarkers. It can also explain the location of putative CD133/CD44-positive CRC CSC in the perivascular niche, which is likely to comprise cancer-associated fibroblasts. The LS1034 in vitro/in vivo model is a valuable tool to unravel the mechanism of stromal-induced CD44v8-10 expression and identify further therapeutically relevant, mutual interrelations between microenvironment and tumorigenic phenotype.


Incubation conditions
Significance

levels (p values) for differences in the engraftment (tumor take rates, TTR) of cell lines (A) and cell line subpopulations defined by their CD44/CD133 surface expression profiles (B-D)
Statistical significance was evaluated by a bootstrapping approach as detailed in the Materials and Methods section; *control = run-through sorter original cells (processed according to the respective subpopulations) (A) Comparison of cell line-specific TTR after injection of 10-10,000 in vitro grown cells (data documented in Figure 1B) (B) Comparison of TTR after injection of 10-100 in vitro grown SW620 cells sorted according to their CD133/CD44 surface expression pattern (data documented in Figure 2B) any CD133/CD44-defined subpopulation n.s.

(C)
Comparison of TTR after injection of 10-100 in vitro grown (CD44-negative) LS1034 cells sorted according to their CD133 surface expression (data documented in Figure 3B) (D) Comparison of TTR after injection of 500-10,000 LS1034 cells derived from xenografts and sorted according to their CD133/CD44 surface expression pattern (data documented in Figure 4B)  Table S1. Notably, the CD133 (AC133) fluorescence signal was enhanced by a two step FASER series as detailed earlier [23,33].   Figure S1A; the right graphs display the data ordered by (i) an increasing SI which quantitatively reflects biomarker surface expression and (ii) the presence of one or two distinct populations.

Figure S3
Flow cytometric dot blot diagrams showing the CD133/CD44 surface pattern in SW480 cell suspensions prepared from three individual xenografts. The samples were pre-gated to exclude non-human and membrane-defect (PI-positive) cells. Aliquots exposed to isotype control antibodies required for analyses of cell fractions are documented in the upper row. For comparison with (i) CD133/CD44 expression in SW480 monolayer culture (cf. Figure 1) and (ii) CD133/CD44 profile in SW620 xenograft cells (cf. Figure 2). Notably, in all SW480 xenograft preparations a minor proportion (0.1-1%) of cells stained with the isotype antibody shows FC signals beyond the major isotype gate limiting the sensitivity of quantitation.

Figure S5
Heterologeous CD44 protein expression in CRC cell lines as detected by Western blotting (WB); 40 µg (A) and 25 µg (C) of total protein was loaded per lane. No WB bands were detected with any of the antibodies and illumination times for LS1034 cells grown under diverse conditions (negative blots not shown). Flow cytometric analyses (FC) reveal CD44 surface presentation on SW620 cells.
(A) Representative WB of CD44 pattern in whole cell protein extracts of various CRC cell lines as detected with the pan-aCD44(1) antibody (Ab); *two independly prepared protein lysates of SW620 monolayer cells (L1, L2) were loaded on this specific SDS-PAGE.
(A) Visualization of the CD44 tv3 and CD44 tv4 transcript structures and density plots showing the expression range in the tissues of interest.
(B) Heatmap of CD44 tv3 and CD44 tv4 transcript-specific expression (% of isoform) in colon adenocarcinoma and primary normal colon epithelium