(J-394) In Living Color: Isosulfan Blue As a Tool to Study Immuno-Engineering In Vivo

Our lab has developed microporous annealed particle scaffolds (MAPS)–injectable biomaterial for in situ immuno-engineering of skin wounds. ¹ We have shown MAPS coordinate adaptive and innate immune cells, providing structural cues to regenerate tensile strength, epidermal morphology and hair follicles. ^2,3 The interplay between MAPS and the immune system led us to explore MAPS-adjunct models of transplantation. A critical component of this work is flow cytometry-based analysis of leukocytes in the lymph nodes (LNs) that drain skin grafts. However, isolation of minuscule murine lymph nodes presents technical challenges that may lead to misidentification, poor cell yield, and unrepresentative data. Adapting tools from surgical oncology, we developed a novel isosulfan blue tracer-based sentinel lymph node biopsy (IB-SLNB) for facile isolation of draining lymph nodes in mice (Figure 1). Identification of sentinel LNs is critical during melanoma and breast cancer surgery; ^4,5 studies have shown that using blue dye enhances a surgeon’s ability to accurately identify LNs. ⁶ Although IB has been used in mouse studies, ⁷ none explored simultaneous multicolor flow cytometry. Our goal was to combine immunology, engineering and surgical principles to develop an easily-taught technique for undergraduate and PhD students that generates reproducible data in mice. We hypothesize IB-SLNB can feasibly be taught to non-clinicians and that blue coloration will not impair multicolor flow cytometric analyses.

Anesthetized mice were injected in the left forefoot with 5-10μL of IB or saline; contralateral limbs were unmanipulated controls. At various time points following injection, animals were euthanized to undergo laparotomy with elevation of flaps to expose the axillary regions (Figure 2). Standardized photography and time-titration were used to optimize visualization of axillary LN (axLN) while minimizing off-target signal. Using a combination of blunt and sharp dissection, bilateral axLN were excised, mechanically processed through 70µm cell strainers (Corning, Corning NY), washed, stained with Trypan blue and enumerated with a Countess III (ThermoFisher, Waltham MA); Bland-Altman was used to test significance. As a second, independent modality, samples were stained with Calcein AM–Ethidium Homodimer III (ThermoFisher), imaged via fluorescent microscopy and analyzed using a custom Java script (Oracle, Austin TX) within Fiji/ImageJ software (NIH, Bethesda MD). ⁸ To assess whether IB affected multicolor flow cytometry, a T cell and antigen-presenting cell immunophenotyping panel was designed to compare samples from IB-treated and untreated regions. Markers included CD3, CD4, CD8, CD44, CD62L, CD45 and Zombie-NIR (details in supplemental Table S1). Cells were acquired via Aurora Northern Lights spectral cytometer (CytekBio, Fremont CA), and analyzed using FlowJo (FlowJo, Ashland OR) and GraphPad (Prism, La Jolla CA). Compositional population differences between IB-treated and untreated samples were assessed with Wilcoxon rank-sum and quantified by the Hodges-Lehmann estimator. ⁹

Lymphatics in the murine dorsolateral flank drain to a number of basins, including axillary and inguinal regions. As drainage patterns can vary, harvest from all regions is important to maximize data from each animal. Though inguinal nodes are easily identified, accessing the axilla is challenging for the novice surgeon, as inadvertent avulsion floods the operative field with blood making accurate lymphatic harvest nearly impossible.

We sought to simplify LN harvest to facilitate flow cytometry-based analyses; axLN were optimally visualized three-to-five minutes after IB injection (Figure 3). At longer intervals, off-target signal accumulated in bladder, bowel and liver, presumably via the vascular system. In human surgery, typically five minutes elapses between IB injection and initial skin incision. When comparing yields (IB vs no-IB), Bland-Altman analysis revealed differences in the number of harvested leukocytes. ¹⁰ On average, IB-SLNB produced higher counts by a factor of 1.36 (see supplemental Figure S1). The use of IB does not impair acquisition of reproducible flow cytometry data: Wilcoxon rank-sum analysis revealed IB- and dye-free SLNB yield T cell populations with statistically similar compositions. Any observable differences in percentages were insignificant: CD4⁺ T cells (50.30% vs. 49.31%, IB-SLNB vs. SLNB; Hodges-Lehmann=1.08%), CD4⁺ central memory T cells (32.91% vs. 32.58%, IB-SLNB vs. SLNB; Hodges-Lehmann=-0.55%), and CD8⁺ T cells(39.16% vs. 43.05%, IB-SLNB vs. SLNB; Hodges-Lehmann=-3.89%) (Figure 4).

IB-SLNB is a powerful tool for isolating leukocytes from anatomic compartments that are relevant for immunologists specializing in transplant, oncology or infectious disease. While SLNB without dyes is possible, it typically requires advanced training to perfect. IB-SLNB is easily taught to undergraduate and PhD graduate students, enabling them to participate more fully in high-impact translational research. Additionally, improved cell yields on a per-animal basis may reduce the number of animals needed per experiment (with ethical and cost ramifications). Involving junior trainees in advanced surgical techniques enhances the impact of all team members, with synergistic effects derived from optimization of specialized roles in the lab. Basic, translational and clinical scientists alike are encouraged to adopt this technique, enhancing the potential of each individual trainee to contribute to the advancement of science.

1.         Griffin DR, Archang MM, Kuan C-H, et al. Activating an adaptive immune response from a hydrogel scaffold imparts regenerative wound healing. Nature Materials. 2021/04/01 2021;20(4):560-569.

2.         Liu Y, Suarez-Arnedo A, Riley L, Miley T, Xia J, Segura T. Spatial Confinement Modulates Macrophage Response in Microporous Annealed Particle (MAP) Scaffolds. Adv Healthc Mater. May 11 2023:e2300823.

3.         Liu Y, Suarez-Arnedo A, Shetty S, et al. A Balance between Pro-Inflammatory and Pro-Reparative Macrophages is Observed in Regenerative D-MAPS. Adv Sci (Weinh). Apr 2023;10(11):e2204882.

4.         Giuliano AE, Ballman KV, McCall L, et al. Effect of Axillary Dissection vs No Axillary Dissection on 10-Year Overall Survival Among Women With Invasive Breast Cancer and Sentinel Node Metastasis: The ACOSOG Z0011 (Alliance) Randomized Clinical Trial. JAMA. 2017;318(10):918-926.

5.         Faries MB, Thompson JF, Cochran AJ, et al. Completion Dissection or Observation for Sentinel-Node Metastasis in Melanoma. New England Journal of Medicine. 2017;376(23):2211-2222.

6.         Boughey JC, Suman VJ, Mittendorf EA, et al. Sentinel lymph node surgery after neoadjuvant chemotherapy in patients with node-positive breast cancer: the ACOSOG Z1071 (Alliance) clinical trial. Jama. Oct 9 2013;310(14):1455-61.

7.         Ye T, He R, Wu Y, Shang L, Wang S. Study on enhanced lymphatic tracing of isosulfan blue injection by influence of osmotic pressure on lymphatic exposure. Drug Dev Ind Pharm. Apr 2018;44(4):535-543.

8.         Suarez-Arnedo A, Torres Figueroa F, Clavijo C, Arbeláez P, Cruz JC, Muñoz-Camargo C. An image J plugin for the high throughput image analysis of in vitro scratch wound healing assays. PLoS One. 2020;15(7):e0232565.

9.         Hodges JL, Lehmann EL. Estimates of Location Based on Rank Tests. The Annals of Mathematical Statistics. 1963;34(2):598-611.

10.       Giavarina D. Understanding Bland Altman analysis. Biochem Med (Zagreb). 2015;25(2):141-51.

11.       David E, Zhu M, Bennett BC, et al. Undernutrition and Hypoleptinemia Modulate Alloimmunity and CMV-specific Viral Immunity in Transplantation. Transplantation. 2021;105(12):2554-2563.