Synopsis

Keywords: Cancer, Tumor, T-cells, macrophages, immunotherapy

Immune cells are major players of the tumor microenvironment (TME), having profound effects on tumor development and metastatic progression. We present oscillating-gradient diffusion-weighted MRI (OGSE-DWI) as non-invasive imaging approach to monitor the intratumoral immune cell infiltrate, relying on size differences between cancer cells, T-cells and macrophages. By applying the Imaging Microstructural Parameters Using Limited Spectrally Edited Diffusion (IMPULSED) model to sine-shaped OGSE-DWI, changes within the TME and its specific immune cell composition were monitored and compared in syngeneic murine breast cancer models with different degrees of malignancy during tumor progression, clodronate liposome-mediated depletion of macrophages and immune checkpoint inhibitor treatment.

Introduction

Immune cells are main players in building, shaping and reprogramming the tumor microenvironment (TME)¹. While the inflammatory TME is characterized by complex and dynamic interconnections between various immune cell subtypes², especially the interaction between T-cells and macrophages has crucial impact on tumor progression and metastatic spread^3,4, so simultaneous imaging of T-cell and macrophage infiltration into tumor lesions is highly desired.
Oscillating-gradient spin-echo diffusion-weighted imaging (OGSE-DWI), combined with the Imaging Microstructural Parameters Using Limited Spectrally Edited Diffusion (IMPULSED) method, enables an accurate quantification of cell sizes⁵. The purpose of this study was to investigate whether OGSE-DWI enables a cell size-based discrimination between cancer cells, T-cells and macrophages, providing a non-invasive and tracer-free imaging approach for distinct in vivo characterization of the inflammatory TME.

Methods

The accuracy of IMPULSED-derived cell radii was evaluated using in vitro spheroid models, consisting of either pure cancer cells, macrophages or T-cells. Subsequently, in vivo experiments aimed to assess changes within the TME and its specific immune cell composition in syngeneic murine breast cancer models with divergent degrees of malignancy (4T1: high malignancy, metastatic, 67NR: low malignancy, non-metastatic) during tumor progression, clodronate liposome-mediated depletion of macrophages and immune checkpoint inhibitor (ICI) treatment. OGSE sequences were performed as multi-shot EPI sequences with 4 segments (TR = 1000 ms, TE = 88 ms, 8 averages, 1.0 mm slice thickness, 18 x 15 mm² FOV, 96 × 64 matrix, three orthogonal diffusion gradient directions, diffusion gradient duration δ = 25 ms, diffusion gradient separation Δ = 30 ms, nine b-values including 0, 0.125, 0.25, 0.375, 0.5, 0.75, 1, 1.5, 2 ms/µm² and gradient frequencies f of 40, 80, 120 and 160 Hz). In contrast to previous studies using cosine-shaped oscillating gradients^5-7, we applied sine-shaped OGSE-DWI to the IMPULSED model to increase the model’s sensitivity for cell radii larger than 10 µm. Cell radii were derived from sine-OGSE diffusion-weighted images with the intracellular signal attenuation β modeled as $$\beta (OGSE_{sine} ) = 2(\gamma g)^2 \sum_n \frac{B_n \omega^2}{(\lambda_n^2 D_{in}^2 + \omega^2)^2} \left\{ \frac{\lambda_n D_{in} \delta (\lambda_n^2D_{in}^2 + \omega^2)}{2\omega^2} + 1 - \exp(-\lambda_n D_{in} \delta) - \exp(-\lambda_n D_{in} \Delta)(1- \cosh(-\lambda_n D_{in} \delta)) \right\}$$ with $$$B_n = \frac{2(r/\mu_n)^2}{\mu_n^2-2}$$$ and $$$\lambda_n = \left(\frac{\mu_n}{r}\right)^2$$$ (γ: gyromagnetic ratio, g: gradient amplitude, D_in: intracellular diffusion coefficient, ω: diffusion gradient angular frequency, B_n and λ_n: structure-dependent coefficients, for IMPULSED with μ_n as the n^th root of $$$\mu J'_{3/2}(\mu) - \frac{1}{2} J_{3/2}(\mu) =0$$$, r: cell radius). To stabilize the fitting procedure and improve signal-to-noise ratios, D_in was set to a fixed value and data were analyzed using an ROI-based approach. Ex vivo validation of IMPULSED-derived cell radii was achieved by immunohistochemical wheat germ agglutinin (WGA) staining of cell membranes, while intratumoral immune cell composition was analyzed by CD3 and F4/80 co-staining. Statistical analysis of in vitro spheroids was performed using two-sided t-test. Longitudinal in vivo data were analyzed by one-way ANOVA with Tukey post-hoc test or Kruskal-Wallis test and cell radii of therapy groups were compared to control groups using two-sided t-test or Mann-Whitney U test (depending on normal distribution of data, checked by Shapiro-Wilk test).

Results

In vitro spheroid experiments validated the capability of OGSE-DWI for a cell size-based discrimination between cancer cells and different immune cells by detecting significantly (p < 0.0001) smaller cell radii for T-cells (3.8 ± 1.8 μm) and significantly (p < 0.0001) larger cell radii for macrophages (13.0 ± 1.7 µm), compared to both cancer cell lines (4T1: 8.8 ± 1.3 μm, 67NR: 8.2 ± 1.4 μm) (Fig. 1). While T-cell infiltration during progression of 4T1 tumors was captured by decreasing IMPULSED-derived cell radii from 9.7 ± 1.0 μm to 5.0 ± 1.5 μm (p < 0.0001) (Fig. 2a-c), increasing amount of intratumoral macrophages during progression of 67NR tumors was detected by increasing mean cell radii from 8.9 ± 1.2 μm to 12.5 ± 1.1 μm (p = 0.0002) (Fig. 2d-f). After macrophage depletion, mean cell radii decreased from 6.3 ± 1.7 μm to 4.4 ± 0.5 μm (p = 0.02). T-cell infiltration after ICI treatment was captured by decreasing mean cell radii in both tumor models three (4T1: 4.6 ± 0.8 μm compared to 6.3 ± 1.7 μm for controls, p = 0.03, 67NR: 7.1 ± 0.5 μm compared to 11.7 ± 0.9 μm for controls, p = 0.003) and six days (4T1: 3.2 ± 0.8 μm compared to 5.0 ± 1.5 μm for controls, p = 0.02, 67NR: 5.2 ± 0.7 μm compared to 12.5 ± 1.1 μm for controls, p = 0.004) after treatment initiation (Fig. 3).

Discussion

This study highlights the capability of non-invasive diffusion-weighted MRI to provide distinct insights into the intratumoral immune cell infiltrate and its composition, by attributing changes and differences in mean cell sizes to the intratumoral T-cell and macrophage content. We have successfully applied sine-shaped OGSE-DWI to precisely distinguish different cellular compositions of the TME, evaluating the effects of tumor progression, macrophage depletion and ICI treatment on averaged cell sizes in murine breast cancer models with different degrees of malignancy.

Conclusion

Sine-shaped OGSE-DWI’s capability to simultaneously assess T-cell and macrophage infiltration into tumor lesions enables non-invasive characterization of the inflammatory TME with the potential to monitor disease progression and early response to immunotherapies.

Acknowledgements

This study was supported by the German Research Foundation (DFG, GE 3336/1-1, grant no. 446302350; SFB1009-A09, -B08 and -Z02, grant no. 194468054), the Interdisciplinary Center for Clinical Research Münster (PIX) and the Medical Faculty of the University of Münster (fellowships to EH, MG). We thank Claudia Terwesten-Solé, Klaudia Niepagenkemper, Richard Holtmeier, Alletta Schmidt-Hederich and Florian Breuer for excellent technical support.