Tumors consist of a heterogeneous mixture of multiple interacting cell types including non-tumorigenic and malignant phenotypes such as tumor cells, cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs) and stromal cells. Only a small subpopulation are cancer cells capable of driving progression and ultimately leading to malignancy. These malignant cells possess unlimited proliferation potential, self-renewal and resistance to cytotoxic drugs. The complexity of tumors makes the purification and characterization of the malignant subpopulation of cancer cells difficult.
In vivo cancer models, including patient derived xenographs (PDX models), have been developed for establishing tumor cultures in living animals since direct in vitro isolation was not effective. After several rounds of serial in vivo transplantation of tumor tissue in severely immune-compromised mice, the cells of the primary tumor eventually develop into a stable tumor cell population. PDX mouse models are believed to conserve the original tumor characteristics including heterogeneous histology, clinical biomolecular signatures, malignant phenotypes and genotypes, tumor architecture and vasculature. However, these techniques are often expensive, time-consuming, elaborate, variable and they induce major changes in the initial primary tumor cells caused by the serial selection process in animals.
Traditional in vitro cancer culture media lack specificity for malignant cells. These media predominately supports the proliferation of benign stromal cells and differentiated cells (non-tumorigenic), thus leading to a gradual loss of the original malignant cell population overtime. The PromoCell Primary Cancer Culture System (C-28081), consisting of Primary Cancer Cell Medium D-ACF (C-28080, C-39880) and the NCCD-Reagent (C-43080), was designed to be the first universally applicable and cost-effective solution for in vitro isolation of long-term primary tumor culture from patient tumor samples or patient derived xenografts (PDX). The selection process uses a proprietary coating NCCD-Reagent and an optimized animal-free cancer cell culture media. The system makes it possible to reliably deplete benign cells from the culture while supporting the maintenance of malignant cancer cells. The system can also be used for other applications such as enriching malignant subpopulation(s) in established cell lines or depleting stromal cells and other non-cancerous cells from established primary cancer cell cultures.
Figure 1. Protocol overview of the selective isolation of primary human cancer cells from patient derived tumor samples or effusions using PromoCell’s Primary Cancer Culture System.
The use of the NCCD-Reagent (C-43080) provided with the Primary Cancer Culture system is required for successful isolation and maintenance of cancer cells. Dilute the thawed NCCD-Reagent stock solution 1:20 with PBS. Use 100 μL/cm2 to treat the tissue culture vessel with the diluted NCCD-Reagent and leave the closed vessel for at least 1 hour at RT. Make sure that the NCDD covers the entire vessel surface. Aspirate the NCCD solution just before seeding the cells.
Figure 2. Appearance of primary culture of lung squamous cell carcinoma cells in the Primary Cancer Cell Medium D-ACF in early stages.A) Culture on day 2 after initial plating: a mix of residual erythrocytes, fibroblast- and epithelial-like adherent cells as well as floating single suspension cells can be observed. B) On day 11, the formation of floating multicellular cell aggregates is already prominent (white arrows). The culture was used for primary aggregate separation on day 13 and was additionally cultured in a new flask parallel to the original sample containing the remaining adherent cell fraction.
Figure 3. Primary culture derived from a low-grade small cell lung cancer with the Primary Cancer Culture System.A) The primary isolate was obtained after 4 weeks as a floating sphere-forming culture, which persisted in a near-quiescent state even after 6 months. B) Adding extra growth factors elicited significant expansion in the latent sphere culture with a doubling time of 3-4 weeks. Note that some spheres persisted under these modified culture conditions (white arrows), while the larger part of the culture proliferated as floating planar multicellular 2D sheets, which is a prototypical growth pattern for SCLC cells in vitro.
Figure 4. Primary culture derived from an invasive adenocarcinoma with the Primary Cancer Culture System. A) During the first two weeks of the isolation process, the cancer cells appeared as locally restricted, lightly-adherent convex cell clusters (white arrow) on top of the stroma layer. B) After 4 weeks, highly motile cancer cells began migrating from their original locations to cover the whole culture surface. These cells proliferated as a homogeneous population in the Primary Cancer Cell Medium D-ACF.
Figure 5. Mutational analysis of the squamous cell carcinoma primary isolate. The tumor panel test detected three hotspot mutations: one in the pik3ca gene and two in tp53. The high mutational load is indicative of a selectively enriched culture of malignant cells, while the differing percentages of the individual mutations suggest the maintenance of cancer cell subpopulation heterogeneity in vitro. Mutational load = percentage of mutated transcripts/total transcripts of the respective transcript variant.
To continue reading please sign in or create an account.Don't Have An Account?