Not all tumors, not even within the same classifications, respond to a treatment in a same way. Personalized or tailored treatment of tumor calls for efficient and reliable assessment of the tumor responsiveness. Even though anatomic and functional imaging have been extensively investigated and applied to assess tumor response to treatment, new biomarkers with sound biological relevance are still needed to assess tumor response to treatment in a time-efficient manner.
In an effort to identify such biomarkers, we previously identify a short peptide (HVGGSSV) with in vivo
phage display technology. The peptide demonstrated potentials in assessing the tumor responsiveness to radiation and tyrosine kinase inhibitors at the early stage of treatment courses 
. As demonstrated within multiple heterotopic and orthotopic tumor models, the peptide selectively binds to the responding tumors, the peptide accumulation within the treated tumors correlates to the overall biological effects of the treatment on the tumor growth control. In this study, TIP-1 was identified as one molecular target of the HVGGSSV peptide. TIP-1 specific antibody competed with the HVGGSSV peptide for binding within irradiated tumors, and exhibited similar binding patterns as the peptide in tumor-bearing mice. It was further identified that radiation induced translocation of the basically intracellular TIP-1 protein onto the cell surface in a dose-dependent manner. The treatment-induced TIP-1 expression on the cell surface is detectable in the first few hours after the treatment and before the onset of treatment associated apoptosis or cell death. In fact, majority of the cells expressing TIP-1 on the cell surface are the live but still responding cancer cells, albeit such cells are less potent in proliferation and more susceptible to subsequent radiation treatment. Although it still under investigation to understand the mechanism and biological consequence of the radiation-induced TIP-1 translocation, these data support one conclusion that the radiation-inducible translocation of TIP-1 onto the cell surface holds promise as one surrogate biomarker in assessing the tumor responsiveness to ionizing radiation.
Discovery of the TIP-1 translocation onto the cell surface as one biomarker of tumor response to radiation took advantages of phage display technologies. Firstly, a peptide HVGGSSV was identified with selective binding to the tumors responding to IR and tyrosine kinase inhibitors with in vivo
phage display 
. Cancer cell distinguishes itself from the normal cell by expressing proteins or receptor on the cell surface, imaging of such surface proteins such as EGFR has been studied to track the tumor progression or even monitor tumor response to treatment 
. We envision that the treatment-inducible protein expression on the cancer cell surface holds promise as surrogate biomarkers for imaging-based assessment of the tumor responsiveness to treatment. Compared to the genomic and proteomic profiling 
that focus on the gene structure and overall expression abundance, in vivo
phage display prefers the molecule that are localized on the cell surface and circulation accessible. Moreover, unlike the subcellular proteomic profiling 
that has been explored in biomarker discovery in vivo
, the in vivo
phage display takes advantages of the minimal sample bias and the real time in vivo
binding within the sophisticated tissues and cell structures. Secondly, TIP-1 was identified as the molecular target of the HVGGSSV peptide through biopanning a phage-displayed cDNA library. In the effort to identify the molecular target(s) of the HVGGSSV peptide, no meaningful data was generated through BLAST search for homologous sequences 
, affinity purification accompanied with mass spectrometric identification 
, or yeast-two hybrid screening of cDNA libraries 
. Low affinity or low abundance of the corresponding molecular target(s) might contribute to the difficulty in identification of the molecular target(s) of the short peptide. A phage-displayed cDNA library was screened against the HVGGSSSV peptide, rounds of biological amplification and affinity selection significantly enriched the peptide-binding clones that lead to the TIP-1 identification. This study further demonstrated the potentials of screening phage-displayed cDNA library in discovery of molecular targets of the peptides with simple structure and low affinity.
TIP-1 is ubiquitously expressed within multiple organs 
. It is predominantly localized in the cytoplasma 
, with rare or undetectable expression in the nucleus or on the cell plasma membrane under normal culturing condition. It has been studied as a PDZ antagonist in modulating cell proliferation, polarity, migration and stress response 
, [ 22]
, [ 26]
, [ 27]
. However, its biological functions in cancer biology and cell stress response are still under investigation. Our flow cytometry and cell imaging data showed that the TIP-1 translocation onto the cell surface after X-ray irradiation was dominantly observed in cancer cells, but did not extend to endothelial cells as tested in this study by the use of HUVEC (). This difference is not related to the abundance of the TIP-1 protein within the cells. Western blot analysis of TIP-1 expression within the whole cell lysates indicated that TIP-1 was expressed in all the cell lines including the HUVEC with comparable protein level (data not shown). Tissue staining also showed that the intravenously administrated TIP-1 antibody was dominantly associated to the tumors cells (). These data suggested that the radiation-induced TIP-1 translocation onto the cell surface might be limited to the tumor cells. This conclusion is also supported by our previous observations that the HVGGSSV peptide did not bind to normal tissues that had been irradiated or inflamed with LPS and TNF-α 
. Contradictorily, radiation-induced translocation of other intracellular proteins such as P-Selectin 
and intercellular adhesion molecule-1 (ICAM-1) 
are reportedly associated with inflammatory response to ionizing radiation and thus not specific to tumor response to the radiation treatment. In this regard, the radiation-induced TIP-1 translocation onto the cancer cell surface is one unique biomarker of tumor response to radiation.
Although further investigation is needed to elucidate the mechanism(s) by which X-ray irradiation induces the TIP-1 translocation onto the cell surface and the biological relevance of the TIP-1 translocation in the tumor response to radiation, we revealed that the cells responding to radiation by relocating TIP-1 onto the cell surface are live but have reduced capability to proliferate and form colonies. The cells with TIP-1 expression on the cell surface are more susceptible to subsequent radiation treatment, compared to the counterparts of the cells without TIP-1 expression on the cell surface. These data partially explains why TIP-1 imaging with the HVGGSSV peptide is predictive in assessing the tumor responsiveness to radiation 
. The TIP-1 translocation was detectable in the first few hours after radiation treatment and before the onset of the treatment associated apoptosis and cell death, suggesting a potential mechanism to assess tumor response to IR at an early time point of a treatment course.