Dept. Biochemistry ; Culture of Primary Cancer Cells and Hypoxia Response

#1 Primary culture of cancer cells: CTOS method

CTOS (cancer tissue-originated spheroid) method was developed in our laboratory (1). The method is for preparation and culture of cancer cells from tumors, based on the principle of maintaining cell-cell contact throughout preparation and culture. Dissociating tumors into single cells has long been a gold standard in primary culture of cancer cells. However most of the cancer cells in solid tumors exist maintaining cell-cell interaction with other tumor cells.

The idea of CTOS method came up with the finding that cancer cells drastically die when they are dissociated into single cells. Therefore we partially dissociate the tumors, filter, and collect the cell clusters upon the filter, discarding the single cells in the flow-through fraction (Figure 1). Another critical finding in the development of CTOS method was that the cell clusters autonomously form spheroids within a short period, few hours. We call these spheroids, which are formed from undissociated clusters, as CTOS. In contrast to the cells dissociated into single-cell, the cells within a CTOS are quite stable (Figure 2), so that cancer cells can be prepared quite efficiently from patient tumors. In addition to the high feasibility, CTOS consists of pure cancer cells. Moreover, CTOS preserves characteristics of original patient tumors in vitro as well as in CTOS-derived xenograft tumors. Thus CTOS provides the opportunity for the study that are clinically relevant.

1. Kondo J, Endo H, Okuyama H, Ishikawa O, Iishi H, Tsujii M, Ohue M, Inoue M. Retaining cell-cell contact enables preparation and culture of spheroids composed of pure primary cancer cells from colorectal cancer. Proc Natl Acad Sci U S A. 2011;108(15):6235-40.

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#2 Hypoxia response of cancer cells

Tumor microenvironment is hypoxic. Cancer cells respond to hypoxia in various ways, which can be classified into three categories: 1) adaptation to hypoxia, 2) renovation of microenvironment, and 3) escape from hypoxia. All of the responses relate to malignancy. Cancer cells adapt to hypoxia by altering metabolism or inducing anti-apoptotic pathway. Hypoxia is one of the strongest inducers of angiogenesis, which resolve the hypoxic microenvironment. Cancer cells in hypoxia escape from deteriorated microenvironment by activating invasion or metastasis. Among these responses, we have been focusing on the adaptation to hypoxia, especially on tumor cell dormancy.

HIF-1a, hypoxia inducible factor-1a, is a transcription factor which plays major roles in the transcriptional regulation in acute hypoxia response. One of the drastic responses is the increase of glucose utilization by activating glycolytic pathway and suppressing pyruvate influx into mitochondria, which are all regulated by the downstream genes of HIF-1a. The paradox in tumor hypoxia is that glucose and other nutrients are also poorly supplied in the region nearby the diffusion limit of oxygen. It is catastrophic for the cancer cells to increase consumption of energy source when the supply is decreased. Therefore, there must be another adaptation mechanism than HIF-dependent responses to acute hypoxia. We are studying tumor cell dormancy as an alternative but critical adaptation mechanism in hypoxia (2).

2. Endo H, Okami J, Okuyama H, Nishizawa Y, Imamura F, Inoue M. The induction of MIG6 under hypoxic conditions is critical for dormancy in primary cultured lung cancer cells with activating EGFR mutations. Oncogene. 2016.

Figure: Hypoxia adaptation

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Location

Department of Biochemistry
Osaka International Cancer Institute
3-1-69 Otemae, Chuo-ku, Osaka, Japan

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