(F-239) Near/Shortwave Infrared Fluorescent Imaging of Vascular Function in the Triple Negative Breast Cancer Tumor Microenvironment with BACH1 Attenuation

Introduction:: BTB and CNC homology1 (BACH1) is a heme binding transcription factor with increased expression in triple negative breast cancer (TNBC) patient tumors. BACH1 expression decreases glucose utilization in aerobic metabolism and has been found to be inversely corelated to electron transport chain (ETC) gene expression (PMID: 30842661). In addition to cellular metabolism, BACH1 has also been shown to play a role in angiogenesis (34482423, 32132179) by modulating vascular endothelial growth factor C (VEGFC). Cell lines expressing a short hairpin RNA for BACH1 can be used to study the effect of depletion of BACH1 on tumor phenotype. The vascularity of tumor microenvironment (TME) plays a large role in tumor growth and therapeutic outcomes, and while tumor cells are relatively well characterized, host vascular phenotypes are not. Tumor vascular perfusion kinetics have been correlated to clinical response to therapy and survival (PMID: 20429756). Dynamic fluorescence imaging with indocyanine green (ICG), which has an absorption and emission spectra that spans the NIR-I and tails into the NIR-II or SWIR (29626132), can be used to quantify host vascular phenotype in a rat xenograft model of TNBC (29552392). This enables it to exploit the higher spatial resolution due to reduced scattering in the SWIR spectrum. Additionally, the impact of BACH1 expression and vascular phenotype on uptake of nanocarrier therapeutics can be imaged in the NIR-I spectrum via DiOC18(7) (DiR) tagged liposomes.


Materials and Methods:: Rats were salt sensitive Sprague-Dawley (SS^IL2Rγ). Xenograft cell lines included high BACH1 expressing MDA-MB-231 (231) and lower BACH1 expressing MDA-MB-436 (436), patient derived triple negative breast cancer cell lines, with a control short hairpin (WT) and shBACH1 (KO) for both cells. The 436 cells also included a mouse BACH1 overexpression line (KI). Animals were implanted with either KO and WT, or KI and WT cell lines on opposite abdominal flanks in the mammary fat pad. For fluorescence imaging, animals were injected intravenously via tail vein with ICG (0.75mg/kg, MP Biomedicals), or 500uL of DiR tagged liposomes and simultaneously imaged. For ICG whole body, an InGaAs focal plane array camera (NIRvana 640ST, Princeton Instruments), equipped with a 25mm lens (Navitar, SWIR-25), with an 1100nm long pass filter (ThorLabs), excited by 808nm diode laser (~5mW/cm²), was used with a frame rate of 10.6 fps for 6 minutes. For DiR liposome imaging, an emICCD camera (PIMAX-4, Princeton instruments), with a 25mm lens (Navitar, DO-25, 0.95 f-stop), with a 785nm holographic notch (HSPF-785.0-2.0, Kaiser Optical System) and 830±nm bandpass filter (830FS10-25, Andover), excited by 785nm diode laser (~0.6 mW/cm²). Respiratory motion correction was performed with MATLAB (MathWorks Inc.). All animal protocols were reviewed and approved by the Medical College of Wisconsin, Institutional Animal Care and Use Committee, where these experiments were performed.

Results, Conclusions, and Discussions:: Previously, SS.BN3^IL2Rγ rats were shown to have greater permeability and retention and subsequently better therapeutic outcomes as compared to SS^IL2Rγ (28567545, 32373218). Therapeutic efficacy could be enhanced if SS rat tumors could be modified in vivo to have the perfusion profile of SS.BN3. BACH1 attenuation has been shown to increase therapeutic efficacy in combination treatments targeting mitochondrial metabolism, however, the impact of BACH1 on tumor vascular phenotype has not been explored in a rat model via dynamic infrared fluorescence imaging. BACH1 overexpression in the moderately BACH1 expressing 436 cells, yields an increase in ICG washout which has previously been correlated to worse therapeutic efficacy, while BACH1 knockout shows a modestly slower washout compared to WT cells. Additionally, much higher liposomal uptake was demonstrated with the attenuation of BACH1 in the higher expressing 231 cells in SS animals, which had been shown to respond much worse to theragnostic nanoparticle treatments. BACH1 attenuation can be used to change the pharmacokinetic perfusion profile of TNBC xenograft tumors in a rat model and thereby sensitize resistant tumor lines to nanocarrier based therapies.


Acknowledgements (Optional): :

References (Optional): :