Association between Tumorigenic Potential and the Fate of Cancer Cells in a Syngeneic Melanoma Model
Gil Z (2013) Association between Tumorigenic Potential and the Fate of Cancer Cells in a Syngeneic Melanoma
Model. PLoS ONE 8(4): e62124. doi:10.1371/journal.pone.0062124
Association between Tumorigenic Potential and the Fate of Cancer Cells in a Syngeneic Melanoma Model
Yakov Krelin
Liron Berkovich
Moran Amit
Ziv Gil
Salvatore V. Pizzo, Duke University Medical Center, United States of America
The self-renewal potential of a cancer cell can be estimated by using particular assays, which include xenotransplantation in immunocompromised animals or culturing in non-adherent serum-free stem-cells media (SCM). However, whether cells with self-renewal potential actually contribute to disease is unknown. Here we investigated the tumorigenic potential and fate of cancer cells in an in-vivo melanoma model. We examined cell lines which were derived from the same parental line: a non-metastatic cell line (K1735/16), a metastatic cell line (K1735/M4) and a cell line which was selected in non-adherent conditions (K1735/16S). All cell lines exhibited similar proliferation kinetics when grown on culture plates. K1735/16 cells grown in soft agar or in suspension non-adherent conditions failed to form colonies or spheroids, whereas the other cell lines showed prominent colonogenicity and spheroid formation capacity. By using sphere limiting dilution analysis (SLDA) in serum-free media, K1735/16S and K1735/M4 cells grown in suspension were capable of forming spheroids even in low frequencies of concentrations, as opposed to K1735/16 cells. The tumorigenic potential of the cell lines was determined in SCID mice using intra footpad injections. Palpable tumors were evident in all mice. In agreement with the in-vitro studies, the K1735/M4 cell line exhibited the highest growth kinetics, followed by the K1735/16S cell line, whereas the K1735/16 cell line had the lowest tumor growth potential (P,0.001). In contrast, when we repeated the experiments in syngeneic C3H/ HeN mice, the K1735/16 cell line produced macroscopic tumors 30-100 days after injection, whereas K1735/M4 and K1735/ 16S derived tumors regressed spontaneously in 90-100% of mice. TUNEL analysis revealed significantly higher number of apoptotic cells in K1735/16S and K1735/M4 cell line-derived tumors compared to K1735/16 tumors (P,0.001). The models we have examined here raised the possibility, that cells with high-tumorigenic activity may be more immunogenic and hence are more susceptible to immune-regulation.
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Funding: This research was supported by the Israel Science Foundation (number 1680/08 and 482/11), the Israel Cancer Association (grant donated by Ellen and
Emanuel Kronitz in memory of Dr Leon Kronitz number 20090068), the Israeli Ministry of Health (number 3-7355), the Weizmann Institute - Sourasky Medical
Center Joint Grant, the Tel Aviv Sourasky Intramural Grant, the ICRF Barbara S. Goodman endowed research career development award (2011-601-BGPC) and a
grant from the US-Israel Binational Science Foundation (number 2007312) to Z.G. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Cancer is a complex disease, involving differences between
tumors or cells within a given tumor, as well as variation between
patients. Within the spectrum of cells in a given tumor,
subpopulations of cells may be phenotypically different and
exhibit distinct proliferative potential. For example, the cancer
stem cell (CSC) model suggests that only a small subpopulation of
cells has a self-renewal and tumor formation potential, while the
majority of the tumor consists of non-tumorigenic cells [1].
Evidence supporting the CSC model is found in germ cell cancer,
leukemia, breast cancer, colon cancer and in some brain cancers.
[2,3,4,5,6,7,8,9,10,11]. On the other hand, whether melanomas
are consistent with such a model is a matter of continuous debate
[12,13].
Currently, the only assay that determines the tumorigenic
potential of human tumors involves xenotransplantation of
different subpopulations of cancer cells into flanks of highly
immunosuppressive animals (e.g. NOD/SCID mice). In addition,
stemness (i.e. the ability to self-renew and differentiate) is
frequently evaluated in-vitro by surrogate assays that examine the
sphere-forming ability and clonogenicity in anchorage
independent conditions, such as semisolid soft agar [14]. Previous
experiments showed that multicellular tumor spheroids are
morphologically and characteristically similar to solid tumours
in-vivo [15,16]. It has also been demonstrated that the
sphereforming potential in suspension non-adherent conditions
consistently correlates with the neoplastic growth potential in
immunosuppressed mice [17,18,19,20].
Both in-vitro and in-vivo stemness assays address the tumorigenic
potential of distinct subpopulation of cells, whereas the actual
formation o (...truncated)