Methods for cancer stem cell (csc) expansion

The method of any one of the preceding claims, wherein said method comprises growing tumoroids from human cancer cells.

The method of any one of the preceding claims, wherein said scaffold comprises an electrospun mixture of poly(lactic-co-glycolic acid) (PLGA) and a block copolymer of polylactic acid (PLA) and monomethoxypolyethylene glycol (mPEG).

The method any one of the preceding claims, wherein said tumoroids are grown in hypoxic conditions or conditions that mimic hypoxic conditions, throughout the cell culture or local to the scaffold.

The method of any one of the preceding claims, wherein said tumoroids are cultured in medium comprising conditioned medium (CM) collected from:

• primary human cancer-associated fibroblasts (CAFs) and/or myeloid-derived suppressor cells (MDSCs) from human peripheral blood.

The method of any one of the preceding claims, wherein said tumoroids are cultured in medium comprising an ECM-based hydrogel.

The method of any one of the preceding claims, wherein a first-generation of said tumoroids have at least a 2-fold, 2.5-fold, or 3-fold increase in CSCs compared to cancer cells used to grow the first-generation tumoroids.

The method of any one of the preceding claims, wherein said method comprises growing a second-generation of tumoroids, and wherein said second-generation of tumoroids have at least a 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold increase in CSCs compared to the first-generation tumoroid cancer cells used to grow the second-generation tumoroids or to the cancer cells used to grow the first-generation tumoroids.

The method of any one of the preceding claims, wherein said method comprises growing a third-generation of tumoroids, and wherein said third-generation of tumoroids have at least a 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 75-fold, or 80-fold increase in CSCs compared to:

• the cancer cells used to grow the first-generation tumoroids, the first-generation tumoroids, or the second-generation tumoroids.

The method of any one of the preceding claims, wherein said CSCs are harvested from a first-, second-, third-, or fourth- fifth-, sixth-, seventh-, eighth-, ninth- or tenth-generation tumoroids.

The method of any one of the preceding claims, wherein said CSCs isolated from said tumoroids are used to grow the next generation of tumoroids.

The method of any one of the preceding claims, wherein said CSCs isolated from first-generation tumoroids are used to grow second-generation tumoroids.

The method of any one of the preceding claims, wherein said CSCs isolated from second-generation tumoroids are used to grow third-generation tumoroids.

The method of any one of the preceding claims, wherein said CSCs isolated from first-generation tumoroids are used to grow second-generation tumoroids and CSCs isolated from said second-generation tumoroids are used to grow third-generation tumoroids.

The method of any one of the preceding claims, wherein said culture or scaffold further comprises one or more iron chelators.

The method of any one of the preceding claims, wherein said culture or scaffold further comprises a siRNA that knocks down von Hippel-Lindau (VHL) tumor suppressor gene.

The method of any one of the preceding claims, wherein said cancer cells comprise a heterologous DNA encoding a growth factor.

The method of any one of the preceding claims, wherein said culture or scaffold further comprises TGF-β.

The method of any one of the preceding claims, wherein said tumoroids are cultured in regular media.