Cell Culture Advances Yield More Reliable Data for Cancer Research

Cells stored with traditional culturing practices express different gene and protein profiles than their in vivo counterparts. Recent studies demonstrate that customizable incubators with settings for oxygen and pressure more accurately recreate the native microenvironment, keeping cells at optimal fitness. Results show that cells stored this way appear molecularly and morphologically similar to their natural state, unlike cells maintained in standard CO2 incubators.

With the increased use of liquid biopsies, improved cell culturing practices are becoming more important. In cancer research, liquid biopsies may target circulating tumor cells (CTCs) found in the bloodstream, using them as prognostic markers. Since these tests are noninvasive and can detect cancer much earlier than other methods, liquid biopsies are of great interest to researchers and clinicians. However, the CTCs recovered from such biopsies have been challenging to study ex vivo. Because their quantities are so limited, many experimental procedures involve enrichment or amplification steps to generate enough DNA or RNA for molecular analysis. These steps introduce bias and sequencing errors that may not accurately reflect the genetic makeup of the tumor.

Ideally, CTCs would instead be cultured, giving them time to divide on their own until the cells naturally created enough genomic material to be analyzed. To date, most CTCs maintained in traditional incubators have failed to propagate. Even if they could be grown with standard culture processes, scientists would doubt the utility of data generated from them, knowing that cell profiles shift significantly once established in incubators.

A new cell culturing technology focuses on carefully reproducing the native microenvironment of cells, with the basic concept that better storage conditions will keep cells functioning as they would in vivo for longer periods of time. The incubator builds on decades of work showing that cells need very specific conditions to thrive. If successful, it would keep cells at optimal fitness, allowing them to grow and divide while maintaining their original gene and protein expression profiles.

Maintaining fitness

Once cells are removed from their native environment, their behavior and expression are altered; they even adopt different morphology. The most basic understanding of how biology works—that DNA makes RNA makes protein—requires an environmental explanation for differentiation of cells into various types when their DNA content is the same.

Breast cancer researcher Mina Bissell, Lawrence Berkeley National Laboratory (Berkeley, Calif.), was one of the first to show that cells lose their tissue-specific function when removed from their native environment and cultured. She also demonstrated that restoring cultured cells to their original home induced them to return to their original traits and behaviors.¹

Two conditions are especially important for maintaining the natural state of a cell: hypoxia and pressure. Johns Hopkins scientist Gregg Semenza made the seminal discovery that key transcription factors are activated by hypoxia. In later studies, his team determined that specific oxygen levels are needed to regulate gene expression activity everywhere from embryonic development to mature adult cells.²

The application of atmospheric pressure and its effects on cells is well understood, but data generated by scientists at Xcell Biosciences (San Francisco, Calif.) suggests that cellular metabolism is significantly altered to promote cell survival. Traditional incubators typically store mammalian cells at 37 °C with 5% CO₂ and 85% humidity. Scientists cannot adjust oxygen or pressure levels, even though there is now a wealth of data supporting the idea that these characteristics are critical to cells’ ability to maintain their identity and fitness. For example, tumor cells in the body are exposed to 1% oxygen and about 1 pounds per square inch of hydrostatic pressure, while immune cells present in the blood experience 10% oxygen and 2 PSI.

Cancer cell studies

Scientists from the Helen Diller Cancer Center at the University of California, San Francisco, Xcell Biosciences and Knowledge Synthesis (Berkeley, Calif.) analyzed CTCs collected from liquid biopsies of patients with late-stage, metastatic castration-resistant prostate cancer. Cells were cultured in an Xcell Avatar bioreactor (Figure 1), which has customizable settings for oxygen and pressure levels to more accurately recreate the native microenvironment.

 Figure 1 – Avatar bioreactor.

Metastatic cell clusters were cultured on a collagen-based cell-binding substrate with 1% oxygen and pressure of 2 PSI, designed to mimic the metastatic niche of the bone marrow. While CTCs usually fail to propagate in traditional incubators, CTCs in this study survived and continued to divide. After seven days, the researchers studied the clusters that had formed colonies with qPCR, immunofluorescence imaging and RNA sequencing.

A quarter of the colonies were tumor cells, identified through biomarker signatures linked to prostate cancer, such as PSMA, EpCAM and cytokeratins. Differential activity was found even in CTC colonies grown from cells taken from the same patient, indicating that cells originated from multiple tumor clones. Of the differentially expressed genes, some were associated with neuroendocrine-like signatures and biomarkers linked to stem cells and immunotherapeutic targets.

The analysis also revealed increased CXCR4-mediated signaling in the cultured cell colonies, which proved to be a critical discovery. In studies of CTCs taken directly from bone marrow, this pathway is consistently elevated and has been considered a strong biomarker candidate for bone metastasis; however, countless analyses of cultured or enriched CTCs have not detected this signaling spike. Scientists have long wondered about this discordance, and these new results suggest that traditionally cultured CTCs have undergone gene expression changes that simply no longer reflect their native biological profile. The fact that this single pathway is so dramatically altered by enrichment or culturing methods calls into question the applicability of other data generated from such cells. Since the profiles of CTCs stored in the customizable incubator reflected the signaling expected from such cells, it appears that adjusting the cell culturing approach may be enough to produce biologically relevant data for cancer-based studies.

In a separate study conducted by scientists at Bayer Healthcare USA (San Francisco, Calif.) and Xcell Biosciences, similar results were found for T cells. Cell-based immunotherapy often involves retrieving T cells from cancer patients, treating them externally to boost their cancer-killing ability and then implanting them back into the patient. In most cases, however, only a small fraction of the restored T cells show any activity against the cancer.

In this study, patient-derived CD8+ T cells were co-cultured with cells from a lung carcinoma cell line. Some were stored in a traditional incubator set at the standard 37 °C and 5% CO₂, and some were stored in the Avatar system with the same CO₂ and temperature settings in addition to 2% oxygen and a pressure level of 2 PSI. Throughout the course of the study, T cell-mediated killing was significantly higher in the Avatar system than in traditional incubators. Based on this data, scientists hypothesized that traditional incubators diminish T cell activity; this would suggest that the incubator-based bioproduction process needed for immunotherapy is actually limiting effects of the T cells when they are reintroduced to the patient. It is possible that storage in more customized conditions during bioproduction would yield T cells with higher activity and therefore increased effectiveness in killing cancer.

Conclusion

Reliable, relevant data from cultured cells will lead to a better understanding of the biological mechanisms involved in cancer formation and metastasis and creative new approaches for treating patients. Better culturing practices will also enable the study of viable rare cells such as CTCs, which are difficult to maintain in traditional incubators.

Advanced cell culturing techniques may make it possible to successfully mimic the metastasis process ex vivo to find potential intervention points. The hope for a truly personalized approach to cancer treatment, where each patient’s cells are cultured for use in assays that will find the most effective therapies, to predict and prevent drug resistance or cancer recurrence, may now come to fruition.

References

1. Bissell, M.J. and LaBarge, M.A. Context, tissue plasticity, and cancer: are tumor stem cells also regulated by the microenvironment? Cancer Cell 2005 Jan, 7(1), 17–23.
2. Iyer, N.V.; Kotch, L.E. et al. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1α. Genes & Dev. 1998, 12, 149–62.

Additional reading

1. Lim, J.; Ryan, C.R. et al. Ex vivo propagation and characterization of circulating tumor cell clusters from late stage prostate cancer patients. Poster presented at Genitourinary Cancers Symposium 2016, San Francisco, Calif.
2. Adams, B.; Lim, J. et al. Characterizing the effects of hypoxia and hydrostatic pressure on the expression of immunotherapeutic targets in prostate cancer cell lines and patient-derived immune cells in culture. Poster presented at AACR 2015, Philadelphia, Penn.

James Lim, Ph.D., is chief scientific officer, Xcell Biosciences, 455 Mission Bay Blvd. South, San Francisco, Calif. 94158, U.S.A.; e-mail: [email protected]; www.xcellbio.com