Abstract Triple-negative breast cancer (TNBC, 15–20% of breast cancers) has poor prognosis, high metastatic potential, and limited treatment options due to the absence of estrogen receptor (ER), progesterone receptor (PR), and HER2 amplification. The tumor microenvironment (TME) plays a key role in driving progression, with inflammatory enzymes such as inducible nitric oxide synthase (NOS2) and cyclooxygenase-2 (COX-2) upregulated in aggressive subtypes. High NOS2 expression in TNBC has been linked to poor overall survival, possibly due to its role in shaping an immunosuppressive microenvironment and activating pro-metastatic signaling cascades. Elevated COX-2 expression is also a hallmark of TNBC, where it facilitates M2 macrophage polarization, regulatory T cell recruitment, and resistance to immune checkpoint blockade. However, their influence on tumor metabolism, lipid accumulation, and collagen dynamics remains unclear. The TME is also spatially heterogeneous, made up of diverse niches that lead to therapy resistance. Microscopy offers insight into these regions, but exogenous dyes hinder translation to live tissues. To overcome this, we employed a label-free, multimodal multiphoton microscopy platform combining second harmonic generation (SHG) for collagen, two-photon autofluorescence (TPAF) for redox metabolism, and coherent anti-Stokes Raman scattering (CARS) for lipid imaging. We examined how NOS2-derived nitric oxide depletion and COX-2 inhibition via indomethacin affect TNBC metabolism, lipid profiles, and collagen architecture. Orthotopic 4T1 TNBC tumors were established in 4 cohorts of BALB/c mice: Group 1 (n=5, wild type (WT) untreated), Group 2 (n=7, WT + indomethacin), Group 3 (n=5, NOS2 knockout (NOS2-/-)), and Group 4 (n=7, NOS2-/- + indomethacin). 12-15 mm diameter tumors were excised, sectioned, and imaged ex vivo. A dual-output ultrafast tunable laser (Insight DeepSee, Santa Clara, USA) was used as the optical source of the label-free multimodal platform. For CARS, TPAF of NADH, TPAF of FAD, and SHG, the following excitation wavelengths were used: 803 nm combined with 1040 nm, 755 nm, 860 nm, and 803 nm. Samples were imaged at 25x magnification (0.94 µm/pixel). Multimodal images of whole slices were acquired and processed using MATLAB. SHG images were quantified by collagen area fraction (CAF), anisotropy parameter, Hough transform-based standard deviation, and Fourier transform-based metrics. Redox ratio was used to assess TPAF images. Size distribution of lipid droplets through CARS was also analyzed. CARS imaging revealed distinct lipid remodeling across both NOS2-NO depletion and indomethacin treatment. NOS2- tumors show a significant increase in large lipid droplets (1,000–10,000 pixels) compared to wild-type, suggesting enhanced lipid accumulation suggesting altered storage or β-oxidation. Indomethacin-treated tumors, independent of NOS2, had a greater share of small lipid droplets (1–10 pixels), reflecting altered lipolysis or turnover. The combined treatment in NOS2- mice further augmented the accumulation of small lipid droplets (p0.0075), supporting a synergistic effect. Global SHG and TPAF analyses did not reveal significant differences, but initial regional analyses are ongoing to assess collagen structure and metabolic redox states in peritumoral and immune-rich zones. Early inspection suggests disorganized collagen in immune dense regions of NOS2- tumors, potentially facilitating immune cell infiltration. Citation Format: W. Poon, R. Uzma, R. Master, L. Wang, H. Zhao, A. Veeraraghavan, L. Ridnour, S. J. Lockett, R. Raghunathan, D. A. Wink, S. T. Wong. Label-free multimodal multiphoton microscopy reveals spatial lipid alterations associated with inflammation in triple-negative breast cancer following NOS2 suppression and COX-2 inhibition abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS5-03-19.
Poon et al. (Tue,) studied this question.