# Enrichment of colon cancer stem cells via polymeric porous filters with different zeta potentials

## TL;DR

This study shows that using filters with specific electrical charges can efficiently enrich colon cancer stem cells, which are crucial for tumor growth.

## Contribution

A novel membrane filtration method using zeta-potential-modified filters to enrich cancer stem cells from colon cancer cells is introduced.

## Key findings

- Positively charged PLG/PLL filters effectively enriched cancer-initiating/stem cells from colon cancer cell suspensions.
- Cells migrating through positively charged filters showed high tumorigenic potential and killed 83% of NOD-SCID mice.
- The method successfully increased CD44 and CD133 marker expression, indicating enrichment of cancer stem cells.

## Abstract

Colorectal cancer is one of the most prevalent malignant tumors worldwide, and cancer-initiating (CI) cells or cancer stem (CS) cells are critical for tumor progression. We purified CI/CS cells from colon cancer cells utilizing a membrane filtration method. We developed membrane filters with different surface charges (zeta potentials) by blending a negatively ionized polymer [poly(vinyl alcohol-itaconic acid), PVI] or a positively ionized polymer [poly(L-lysine), PLL] into poly(lactide-co-glycolic acid) (PLG). Suspensions of HT-29 colon cancer cells and PAT-3 cells (primary colon cancer cells from a colon cancer patient in this research) were permeated through unmodified PLG filters and modified PLG filters blended with and without PVI or PLL. The cells in the filtration and recovering solutions and migrating cells from the filters after filtration were evaluated to identify the cells in each fraction and which filter enriched CI/CS cells. CI/CS cells were evaluated for (a) CD44 and CD133 (CI/CS cell markers) expression by flow cytometry and immunostaining in vitro. Cells were also (b) evaluated by a colony formation assay in vitro, (c) evaluated for carcinoembryonic antigen (CEA) production by enzyme-linked immunosorbent assay in vitro and (d) evaluated for a xenograft tumorigenicity test using NOD.CB17-Prkdcscid/NcrCrl (NOD-SCID) mice in vivo. The results revealed that the migration of colon cancer cells from positively charged PLG/PLL filters increased the number of CI/CS cells efficiently and killed 83% of NOD-SCID mice after transplantation, whereas the other fraction of cells did not kill NOD-SCID mice. The filtration method through PLG/PLL filters is effective for purifying CI/CS cells from colon cancer cells.

Graphical Abstract