Intratumor Heterogeneity and Treatment Resistance of Solid Tumors with a Focus on Polyploid/Senescent Giant Cancer Cells (PGCCs)

Abstract

Single cell biology has revealed that solid tumors and tumor-derived cell lines typically contain subpopulations of cancer cells that are readily distinguishable from the bulk of cancer cells by virtue of their enormous size. Such cells with a highly enlarged nucleus, multiple nuclei, and/or multiple micronuclei are often referred to as polyploid giant cancer cells (PGCCs), and may exhibit features of senescence. PGCCs may enter a dormant phase (active sleep) after they are formed, but a subset remain viable, secrete growth promoting factors, and can give rise to therapy resistant and tumor repopulating progeny. Here we will briefly discuss the prevalence and prognostic value of PGCCs across different cancer types, the current understanding of the mechanisms of their formation and fate, and possible reasons why these tumor repopulating "monsters" continue to be ignored in most cancer therapy-related preclinical studies. In addition to PGCCs, other subpopulations of cancer cells within a solid tumor (such as oncogenic caspase 3-activated cancer cells and drug-tolerant persister cancer cells) can also contribute to therapy resistance and pose major challenges to the delivery of cancer therapy.

Keywords: anastasis; apoptosis; cancer therapy; intratumor heterogeneity; polyploid giant cancer cells; precision oncology; preclinical assays; senescence.

Figure 1

Complex heterogeneity within an individual solid tumor (adapted from [8]).

Figure 2

Fluorescence images showing the morphology of MDA-MB-231 cells before (control) and after treatment with the indicated drugs for 3 days. Reproduced from Mirzayans et al. [32].

Figure 3

Cartoon illustrating the generation and fate of polyploid/senescent giant cancer cells (PGCCs). Anticancer treatment triggers the creation of PGCCs that often enter a state of dormancy (active sleep) and thus might be overlooked or scored as “dead” in conventional preclinical assays. A subset of PGCCs, however, remain viable, secrete growth promoting factors, and can give rise to therapy resistant and tumor repopulating progeny through neosis (nuclear budding and bursting), depolyploidization involving meiosis and self-renewal genes, and sub-genome transmission (transfer of nuclear material into surrounding cells via cytoplasmic tunnels). For further details, see [20].

Figure 4

Phase-contrast microscopy images showing features of senescence in the indicated breast cancer cell lines. Cells were exposed to ionizing radiation (8 Gy) or sham-irradiated (control), incubated for seven days, and evaluated for morphology and positive (blue) staining in the senescence-associated β-galactosidase (SA β-Gal) assay. Some regions containing “small-sized” SA β-Gal-positive cells are marked. Reproduced from Mirzayans et al. [51].

Figure 5

(A) Bright-field microscopy images showing the ability of MDA-MB-231 cells to convert the MTT reagent to its formazan metabolite (dark granules and crystals) before (control) and after incubation with cisplatin (10 µM), oxaliplatin (10 µM), or paclitaxel (20 nM) for 3 days. Images were acquired after incubation of cells with MTT for ~1 h. (B) Percentages of polyploid/senescent giant cells and MTT-positive cells in cultures of the MDA-MB-231 cell line before (control) and after treatment with cisplatin (10 μM), oxaliplatin (10 μM), or paclitaxel (20 nM) for 3 days. Only adherent cells were evaluated. Bars, standard error (SE). (C) Effect of cisplatin treatment (3 days) on the extent of cell proliferation (determined by the direct cell counting assay) and cell membrane integrity (determined by the trypan blue-exclusion assay). Bars, SE. TB, trypan blue. (D) Response of MDA-MB-231 cells to cisplatin (3-day incubation with the indicated concentrations), evaluated by the 96-well plate XTT (solid squares) and CellTiter-Blue (open squares) “viability” assays. These images and data are reproduced from Mirzayans et al. [32,55].

Figure 6

Cartoon illustrating the dark side of apoptosis. Cancer cells with molecular, biochemical, and morphological features of apoptosis are capable of promoting tumor repopulation via different routes, including: (i) secretion of pro-survival factors that is regulated by caspase 3 and involves various signaling pathways, including JNK (c-Jun N-terminal kinase); and (ii) the ability to return from the brink of apoptotic death, resulting in the emergence of progeny with increased numbers of micronuclei and chromosomal abnormalities that can lead to increased aneuploidy, a driving force of aggressive cancer (reviewed in [17]). These various oncogenic functions associated with “dying” (apoptotic) cancer cells include phoenix rising, failed apoptosis, and anastasis. “Treacherous apoptosis” refers to regions within a tumor that are enriched with caspase 3-positive cells (see text for details).

Figure 7

(A) A phase-contrast microscopy image reproduced from the original work of Puck and Marcus that was published in 1956 [71], reporting the effect of ionizing radiation (9 Gy) on the colony-forming ability of HeLa cell cultures. The image shows remarkable (~10 times) size differences between proliferating (colony forming) cells and proliferation arrested polyploid giant cells. (B) Data obtained by us [54] with HeLa and the indicated cell lines that were exposed to ionizing radiation and incubated for 3 days.