Background

Dysregulation of cell cycle is one of the most important hallmarks of cancer. Cell cycle progression is driven by the phasic expression of cyclins and activation of cyclin-dependent kinases. Given that the CDK4/6-Rb axis is essential for the transition from G1 to S phase in the cell cycle, it is a popular drug target in several types of cancer. Although CDK4/6 inhibitors can effectively improve the prognosis of HR⁺/HER2^– breast cancer, there are differences in the sensitivity to CDK4/6 inhibitors among individuals. Some patients show de novo resistance, while others acquire resistance to CDK4/6 inhibitors after a few cycles of therapy. There is thus an urgent need for a precise method to assess the therapeutic response to CDK4/6 inhibitors and to differentiate who is likely to be sensitive to those drugs.

Methods

The HSA-IP nanocomplexes were obtained by conjugated Palbociclib with ICG-NHS, and blended them with human serum albumin (HSA) through self-assembly. Cellular uptake and site-blocking experiments were conducted to evaluate the targeting ability of HSA-IP. To study the ability of HSA-IP to assess the CDK4/6 kinase activity in living cells, MCF-7 was pre-treated with different siRNA or Palbociclib and detected the change of the expression level of pRb, cell cycle distribution and mean fluorescence intensity (MFI) of HSA-IP. The targeting ability of HSA-IP in vivo was investigated by injecting the MCF-7 tumor-bearing mice with HSA-ICG or HSA-IP or 50-fold excess palbociclib followed by HSA-IP and imaging them in NIR-II imaging system. To study whether HSA-IP could assess the response to CDK4/6 inhibitor, we subjected the MCF-7 tumor-bearing mice to daily treatment with 150 mg/kg Palbociclib for 7 days. NIR-Ⅱ imaging before and after treatment were conducted to obtain the image information about CDK4/6 kinase activity. Finally, biosafety of HSA-IP was evaluated.

Results

HSA-IP could be taken up more by cells than ICG or IP or HSA-ICG, and the MFI could be reduced significantly by blocking with Palbociclib (39.37%, P < 0.0001). We treated the MCF-7 with siRNA targeting CDK4 or CDK6 or CyclinD1 or CDK4/6-CyclinD1 complex, leading to a partial reduction of pRb (S795) level and G1 phase block in varying degrees in different groups. The MFI of the above cells with HSA-IP showed a corresponding decrease. Palbociclib caused a dose-dependent decrease in pRb level, and as the concentration of Palbociclib increased, up to 79.83% of cells were arrested in G1 phase. When we applied HSA-IP to visualize the inhibition of CDK4/6 kinase activity, we found that the does-dependent reduction of MFI was coincided with the decrease of pRb. The fluorescence signal of MCF-7 mice injected with HSA-IP intensified primarily in the tumor, and the intensity could be significantly reduced by blocking with Palbociclib (29.30%, P < 0.01). Subsequently, after a 7-day treatment with Palbociclib to MCF-7 tumor-bearing mice, we found there were clear reductions in the MFI of tumor and percentage of cells positive for Ki67 and pRb in tumor tissue between baseline and day 7, even the volume of tumors did not decrease, demonstrating that HSA-IP can assess the antiproliferative effect of Palbociclib at an early stage of treatment in a non-invasive way. Last but not least, HSA-IP exhibited good biosafety.

Conclusions

In this study, we developed a novel probe by clinically accessible materials and succeeded in using it to assess the cancer-associated CDK4/6 kinase activity and the therapeutic response to the CDK4/6 inhibitor in living cells and in vivo. In view of its performance in providing functional information about kinase activity, HSA-IP may be promising as an alternative approach for analyzing morphology and histology of clinical cases, as well as for drug development.

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