Fig 1.
KISS1 and KISS1R are variably expressed in MSCs, OPCs, and tumor cell lines.
Protein expression of the kisspeptin precursor KISS1 and the receptor KISS1R was investigated in primary MSCs (A) and OPCs (B). Specificity of the KISS1R antibody was confirmed by the addition of a blocking antigen (C). Actin was used as a loading control. Dividing lines indicate samples, which were loaded on different gels.
Fig 2.
Direct co-culturing with INA-6 cells significantly upregulates KISS1R expression in MSCs and OPCs.
KISS1R expression analyses of MSCs and OPCs after direct co-culturing with the MM cell line INA-6 (A). Both MSCs and OPCs express significantly higher KISS1R levels (relative to EEF1A1 controls) in response to direct contact with MM cells (n = 5) (B). Up-regulation of KISS1R expression in MSCs is mainly contact-dependent, since indirect trans-well co-culturing techniques could not significantly elevate KISS1R expression (n = 3) (C). Graphs represent average values ± SD. (*p<0.05, ***p<0.001). Dividing lines indicate if samples were loaded on different gels.
Fig 3.
The fluorescent dye Alexa 633 successfully conjugates to the kisspeptin after maleimide-based reaction.
HPLC confirmed the successful conjugation of kisspeptin with Alexa 633. Generation of the Alexa 633-kisspeptin probe results in a notable shift in elution time, measured at both 633 nm (A) and 280 nm (B) with no detectable unlabeled peptide. Elution peaks at both wavelengths are identical (C), indicating successful conjugation of the Alexa 633 dye and kisspeptin.
Fig 4.
Alexa 633-kisspeptin binding to MSCs increases after direct co-culture with either human or mouse-derived MM cells.
Primary MSCs from two different donors were incubated with Alexa 633-kisspeptin or the unconjugated dye and imaged with fluorescence microscopy (A). Dye shows heterogeneous, low level binding to MSCs in a kisspeptin-dependent manner. MSCs were incubated for 24 h with either MM cell line-derived media (conditioned media) or were directly co-cultured for 24 h with INA-6 cells or MOPC cells (B). Binding of the Alexa 633-kisspeptin probe required direct co-culture, while the free dye failed to show any specific binding. Bar = 50μm.
Fig 5.
Alexa 633-kisspeptin binds to tumor-burdened limbs with increased peak binding and altered binding kinetics.
Alexa 633-kisspeptin was injected into immune competent BALB/c mice and its biodistribution determined using the IVIS Spectrum (A-B). BALB/c mice intravenously-injected with the syngeneic MM cell line MOPC were monitored for tumor growth with bioluminescence imaging. Upon tumor development, mice were subsequently injected with Alexa 633-kisspeptin (C). Overall fluorescence of tumor-bearing limbs and their contralateral control limbs, relative to fluorescence detected in the pre-scan, were determined over time (D). Tumor-bearing limbs showed significantly increased relative peak fluorescence (E). Graphs represent average values ± SD. (n = 6) (**p<0.01).