Isolation and Characterization of Cervical Cancer-Associated Mesenchymal Stem Cells From Primary Tumors Using Explant Culture

Abstract

Cancer-associated mesenchymal stem cells (Ca-MSCs), an integral part of the tumor microenvironment, play a major role in modulating tumor progression; they have been reported to progress as well as inhibit various cancers, including cervical cancer. To understand the exact role of Ca-MSCs in tumor modulation, it is necessary to have an optimized protocol for Ca-MSCs isolation. This work demonstrates the isolation and expansion of a primary culture of cervical cancer-associated MSCs (CCa-MSCs) from the biopsy sample of cervical cancer patients using the explant culture technique. The isolated cells were characterized according to International Society for Cellular Therapy (ISCT) guidelines. Morphological analysis revealed that cells were adherent to the plastic surface and possessed spindle-shaped morphology. Flow cytometry analysis of the cells showed high expression (~98%) for MSC-specific cell surface markers (CD90, CD73, and CD105), negative expression (<0.5%) for endothelial cell marker (CD34) and hematopoietic cell marker (CD45), and negligible expression for HLA-DR, as recommended by ISCT. Further, trilineage differentiation potential analysis of the cells showed their osteogenic and chondrogenic potential and adipogenic differentiation. This standardized protocol will assist in the cultivation of CCa-MSCs and the study of their interactions with tumor cells and other components of the tumor microenvironment. This protocol may be utilized in the establishment of Ca-MSCs from other types of cancers as well. Key features • Isolation and expansion of cervical cancer-associated mesenchymal stem cells (CCa-MSCs) from patient biopsy sample. • Characterization of isolated CCa-MSCs for the presence of MSC-specific cell surface markers and trilineage differentiation potential. • CCa-MSCs can be employed to study the interactions with the tumor cells in the tumor microenvironment. Graphical overview.

Keywords: Adipogenic; Cancer stem cells; Cancer-associated MSCs; Cervical cancer; Chondrogenic; Explant culture; Mesenchymal stem cells; Osteogenic; Trilineage differentiation; Tumor microenvironment.

Figure 1.. Stepwise protocol for establishing explant culture.

(i) A biopsy sample was brought to the laboratory in PBS, (ii) washed with ice-cold PBS 3–4 times, and (iii) dissected into small pieces. (iv) 5–6 pieces were placed in each well of a 6-well culture plate.

Figure 2.. Primary culture of cervical cancer–associated MSCs (CCa-MSCs).

(i) A single explant attached to the surface of the culture plate. (ii) Adherent spindle-shaped cells migrating out of the explants on Day 3. (iii) The explant surrounded by more adherent cells by Day 9. (iv) Spindle-shaped morphology of CCa-MSCs at passage 2. Scale bar = 100 μm.

Figure 3.. Characterization of cervical cancer–associated MSCs (CCa-MSCs) for cell surface markers.

Characterization of cell surface markers by flow cytometry shows positive expression of MSC-specific surface markers (CD73, CD105, and CD90) and negative expression for hematopoietic cell marker (CD45), endothelial cell marker (CD34), and HLA-DR. The experiment was done in duplicates on three different patient samples.

Figure 4.. Trilineage differentiation of cervical cancer–associated MSCs (CCa-MSCs).

Brightfield microscopic images showing the trilineage differentiation of isolated CCa-MSCs as depicted by uptake of (i) Oil red O, (ii) alizarin red S, and (iii) alcian blue stain by adipocytes, osteocytes, and chondrocytes, respectively. Scale bar = 100 μm (i), 200 μm (ii), and 400 μm (iii). Each experiment was done in triplicate on three different patient samples.