Development of Sentinel LN Imaging with a Combination of HAase Based on a Comprehensive Analysis of the Intra-lymphatic Kinetics of LPs

Abstract

The sentinel lymph node (LN) is the first LN to which lymph fluid flows from tumor tissue. We identified the key parameters of liposomes (LPs) that affect their accumulation in regional (primary) LNs with minimum leakage to its connecting (secondary) LNs by a comprehensive analysis of the LN-to-LN trafficking of LPs with various surface charges and various sizes. We used a lymphatic flow-modified (LFM) mouse that allows for the chronological analysis of inguinal (primary) LN-to-axillary (secondary) LN at the body surface. As a result, the anionic medium-sized LPs (130 nm on average) exhibited the highest accumulation in the primary LNs. A mechanism-based analysis revealed that CD169-positive macrophages in LNs were the dominant cell population that captures anionic LPs. Sentinel LN imaging was also performed by the intratumoral injection of fluorescent medium-sized anionic LPs using a breast cancer orthotopic model. In comparison with the typically used contrast agent indocyanine green, the anionic LPs were detected in sentinel LNs with a high sensitivity. Additionally, the co-injection of hyaluronidase significantly improved the sensitivity of detection of the fluorescent LPs in sentinel LNs. In conclusion, medium-sized anionic LPs combined with hyaluronidase represents a potent strategy for investigating sentinel LNs.

Optimization of Sentinel Lymph Node Imaging Methodology Using Anionic Liposome and Hyaluronidase

“…There are three types of macrophages in LN: subcapsular sinus macrophage (SSM), marginal sinus macrophage (MSM), and marginal cord macrophage (MCM). In the previous report, we have also shown that uptake especially by MSM and SSM of these three types of macrophages is important for the retention in LNs [15].…”

“…PS is reported to have a high affinity to macrophages via the T cell immunoglobulin and mucin domain-containing protein 4 (Tim4) [22,23]. As already mentioned, in previous reports we showed that liposome uptake by macrophages is a crucial factor for the retention of liposomes in LN [15]. Therefore, we hypothesized that PS may have a higher affinity for macrophages among anionic lipids, and hence increased accumulation to LN.…”

“…Our previous reports and non-published data have shown that the incorporation of negatively charged lipids (i.e., phosphatidic acid and phosphatidylglycerol), was not superior to that of LN accumulation of CHEMS liposomes. This suggests that physicochemical property (anionic charge) cannot simply explain the extensive uptake of PS-containing liposomes to the macrophages [15]. We speculate that PS liposomes are taken up by macrophages and lodged in lymph nodes using not only the affinity of the anionic nature itself but also the affinity of the PS receptor.…”

“…Furthermore, using this LFM model, we comprehensively analyzed the effect of physical properties (size, surface potential) of liposomes on their kinetics in the lymphatic system. As a result, we found that anionic liposomes with cholesteryl hemisuccinate (CHEMS) with 130 nm diameter well accumulated in the primary LNs, and retained in it [15]. This property was applicable to the imaging of the sentinel lymph nodes using tumor-bearing mice.…”

“…The LN in the inguinal meridian closest to the transplant site was considered the SLN. HAase (2500 U) was administered into tumor tissue, whose amount was determined based on our and other groups' previous reports [15,19,20]. The experimental protocols were reviewed and approved by the Chiba University Animal Care Committee in accordance with the Guide for Care and Use of Laboratory Animals (Dou3-253).…”