Demonstrating preclinical efficacy of a targeted CDK4 inhibitor by image cytometry.

Cancer researchers continuously search for methods to selectively kill cancer cells with as few adverse side-effects for the patient as possible. A time-tested approach to treating cancer is the use of chemotherapy, small molecules that target rapidly dividing cancer cells by inflicting DNA damage.

Unfortunately, these chemotherapies also target rapidly dividing healthy cells, negatively impacting the health of the patient. As an alternative approach, scientists have discovered ways to target signaling pathways that promote cancer cell growth instead.

The cyclin-dependent kinases, CDK4 and CDK6, are components of a signaling pathway that is critical for promoting cell proliferation in many cancers. CDK4 and CDK6 accomplish this by phosphorylating and inactivating retinoblastoma protein, RB1, and RB1-like proteins that act as transcription inhibitors. This action triggers a series of gene expression changes that drive cell division. Therefore, blocking CDK4 and CDK6 activity offers a more targeted approach to cancer treatment.¹

In a notable recent study, Palmer et al.² address a problem with breast cancer therapies that target the CDK4/6 signaling pathway. They developed a more effective CDK inhibitor and provided preclinical evidence for its efficacy. Important support for the study’s conclusions comes from image cytometry experiments that show the inhibitor blocks cancer cell proliferation in vitro.

Using image cytometry to demonstrate targeted CDK4 inhibitor efficacy

As described by Palmer et al.,² hormone receptor-positive, HER2-negative breast cancers are highly dependent on CDK4 signaling. Current standard-of-care treatments that take advantage of this dependency are dual CDK4/6 inhibitors that also target CDK6. While breast cancer does not rely on CDK6, CDK6 nevertheless remains important for hematopoiesis, and treatment with dual CDK4/6 inhibitors causes neutropenia. To address this limitation, Palmer et al.² sought to generate a CDK4-specific inhibitor, atirmociclib, and evaluate its activity in established in vitro models.

The ability of atirmociclib to inhibit cancer cell proliferation was demonstrated through a viability assay using cultured breast cancer cell lines. The ZR751 cancer cell line was treated for five days with atirmociclib or palbociclib, one of the current standard-of-care dual CDK4/6 inhibitors. At the end of the experiment, viable cells were fluorescently stained and counted on the Celigo™ image cytometer Celigo Image Cytometer .

By testing a wide range of concentrations, strong inhibition of cell growth was observed. This decrease in proliferation correlated with a decrease in RB1 phosphorylation, which is consistent with the proposed mechanism of action for atirmociclib.

To further build their case for atirmociclib, Palmer et al.² showed it inhibits growth of tumor spheroids derived from cancer cell lines, providing a more physiologically relevant three-dimensional preclinical model. To create spheroids, cells from the T47D, BT474, and LNCAP cell lines were centrifuged together in an ultralow attachment plate, and they were treated with atirmociclib or palbociclib after they had formed into a spheroid.

At the tested clinically relevant concentrations of 300 nM or 1000 nM, atirmociclib inhibited the growth of the spheroids more strongly than palbociclib at its clinically relevant concentration of 30 nM.

The current standard-of-care for hormone receptor-positive, HER2-negative breast cancer is to employ both dual CDK4/6 inhibitors and endocrine therapy targeting the estrogen receptor.² In the spheroid model, the combination of atirmociclib with the endocrine therapy fulvestrant led to the strongest inhibition of growth when compared to either treatment individually.

Together with numerous other experiments, these data suggest that atirmociclib could potentially function within the current therapeutic paradigm for treating hormone receptor-positive, HER2-negative breast cancer.

Conclusion

In the experiments above, Palmer et al.² rely on Celigo image cytometry Celigo Image Cytometer  to demonstrate that atirmociclib inhibits cancer cell growth across multiple preclinical models, including cell cultures and 3D spheroids.

The high-throughput analysis capabilities of the Celigo image cytometer allowed the testing of multiple cell lines under many different conditions and it’s rapid imaging and analysis capabilities can scale to the needs of diverse preclinical research workflows, making it an essential tool for many researchers.