| Author: | Yang, Minfeng |
| Title: | Label-free metabolic imaging for sensitive and robust monitoring of anti-CD47 immunotherapy response in triple-negative breast cancer |
| Advisors: | Yoo, Jung Sun (HTI) Cheng, Kenneth (HTI) |
| Degree: | Ph.D. |
| Year: | 2023 |
| Department: | Department of Health Technology and Informatics |
| Pages: | xxiii, 163 pages : color illustrations |
| Language: | English |
| Abstract: | Background Immunotherapy is now revolutionizing cancer therapy from traditional chemotherapies and radiotherapies to immune checkpoint blockades, which utilize patients’ own immune systems to attack and kill tumor cells. Despite the vigorous development of immunotherapies and huge clinical success, there remains an urgent unmet need for a powerful tool to identify responders to different immunotherapeutic modalities. Methods Here, we developed a label-free metabolic intravital imaging technique (LMII) to detect autofluorescence from Reduced Nicotinamide Adenine Dinucleotide Phosphate Hydrogen (NAD(P)H) together with Flavin Adenine Dinucleotide (FAD), two major coenzymes, as powerful imaging markers to consistently monitor metabolic responses to immune checkpoint blockade. The 4T1_PB3R murine breast cancer cell line transduced by a monomeric red fluorescent protein (RFP) gene was utilized for the establishment of skinfold chamber models in female BALB/C mice. These animals subsequently received anti-CD47 immunotherapy (MIAP301), chemotherapy, or radiotherapy, and the systemic therapeutic responses were evaluated utilizing the imaging modality established here. Results First, we investigated the main mechanisms of the CD47-SIRPα pathway to illustrate the metabolic phenotypes of 4T1-PB3R+ cells and macrophages, and to unravel the phenomenon of macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) in vivo. We further visualized a significant enhancement in the autofluorescence of NAD(P)H and FAD during tumor growth. Following MIAP301 immunotherapy, the NAD(P)H and FAD imaging signals were dramatically decreased, verifying the high potential of this monitoring approach for immunotherapeutic responses. NAD(P)H and FAD optical metabolic imaging also revealed a notable decrease after radiotherapy or chemotherapy. Comparative studies with 2-DG 750 fluorescent glucose imaging and traditional bioluminescence imaging verified the high sensitivity of LMII. A validation study was performed by flow cytometry to support the metabolic imaging data. Finally, in vivo immunofluorescence indicated that NAD(P)H imaging mostly targeted cancer cells and a tiny fraction of immuno-active cells, while FAD imaging primarily targeted immunosuppressive populations. Conclusions In summary, our study documents that the well-established LMII technique has a potential as a reliable tool for visualizing heterogeneous cell metabolic characteristics of immunological infiltrates and tumor cells in response to immunotherapy, therefore offering extensive and sensitive surveillance. Overall, the reported imaging technique based on the combination of cellular-level analyses and optical intravital metabolic imaging provides particularly valuable imaging biomarkers for emerging immune checkpoint inhibitors (ICIs) such as anti-CD47 therapy, thus providing new insights into tumor heterogeneity and future clinical tries. |
| Rights: | All rights reserved |
| Access: | open access |
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