Metastatic lymph node targeted CTLA4 blockade: a potent intervention for local and distant metastases with minimal ICI-induced pneumonia.

BACKGROUND: Immune checkpoint blockade (ICB) elicits a strong and durable therapeutic response, but its application is limited by disparate responses and its associated immune-related adverse events (irAEs). Previously, in a murine model of lymph node (LN) metastasis, we showed that intranodal administration of chemotherapeutic agents using a lymphatic drug delivery system (LDDS) elicits stronger therapeutic responses in comparison to systemic drug delivery approaches, while minimizing systemic toxicity, due to its improved pharmacokinetic profile at the intended site. Importantly, the LN is a reservoir of immunotherapeutic targets. We therefore hypothesized that metastatic LN-targeted ICB can amplify anti-tumor response and uncouple it from ICB-induced irAEs. METHODS: To test our hypothesis, models of LN and distant metastases were established with luciferase expressing LM8 cells in MXH10/Mo-lpr/lpr mice, a recombinant inbred strain of mice capable of recapitulating ICB-induced interstitial pneumonia. This model was used to interrogate ICB-associated therapeutic response and immune related adverse events (irAEs) by in vivo imaging, high-frequency ultrasound imaging and histopathology. qPCR and flowcytometry were utilized to uncover the mediators of anti-tumor immunity. RESULTS: Tumor-bearing LN (tbLN)-directed CTLA4 blockade generated robust anti-tumor response against local and systemic metastases, thereby improving survival. The anti-tumor effects were accompanied by an upregulation of effector CD8T cells in the tumor-microenvironment and periphery. In comparison, non-specific CTLA4 blockade was found to elicit weaker anti-tumor effect and exacerbated ICI-induced irAEs, especially interstitial pneumonia. Together these data highlight the importance of tbLN-targeted checkpoint blockade for efficacious response. CONCLUSIONS: Intranodal delivery of immune checkpoint inhibitors to metastatic LN can potentiate therapeutic response while minimizing irAEs stemming from systemic lowering of immune activation threshold.

Drug formulation augments the therapeutic response of carboplatin administered through a lymphatic drug delivery system.

Treatment of metastatic lymph nodes (LNs) is challenging due to their unique architecture and biophysical traits. Systemic chemotherapy fails to impede tumor progression in LNs due to poor drug uptake and retention by LNs, resulting in fatal systemic metastasis. To effectively treat LN metastasis, achieving specific and prolonged retention of chemotherapy drugs in the tumor-draining LNs is essential. The lymphatic drug-delivery system (LDDS) is an ultrasound-guided drug-delivery methodology for administration of drugs to LNs that addresses these requirements. However, early-stage metastatic LNs have an additional set of drug transport barriers, such as elevated intranodal pressure and viscosity, that negatively impact drug diffusion. In the present study, using formulations of elevated osmotic pressure and viscosity relative to saline, we sought to favorably alter the LN's physical environment and study its impact on pharmacokinetics and consequently the therapeutic efficacy of carboplatin delivered using the LDDS. Our study confirmed the capability of a drug formulation with elevated osmotic pressure and viscosity to alter the architecture of LNs, as it caused notable expansion of the lymphatic sinus. Additionally, the study delineated an optimal range of osmotic pressure and viscosity, centered around 1897 kPa and 11.5 mPa·s, above and below which therapeutic efficacy was found to decline markedly. These findings suggest that formulation osmotic pressure and viscosity are parameters that require critical consideration as they can both hinder and promote tumorigenesis. The facile formulation reported here has wide-ranging applicability across cancer spectrums and is thus anticipated to be of great clinical benefit.

Characterizing perfusion defects in metastatic lymph nodes at an early stage using high-frequency ultrasound and micro-CT imaging.

A perfusion defect in a metastatic lymph node (LN) can be visualized as a localized area of low contrast on contrast-enhanced CT, MRI or ultrasound images. Hypotheses for perfusion defects include abnormal hemodynamics in neovascular vessels or a decrease in blood flow in pre-existing blood vessels in the parenchyma due to compression by LN tumor growth. However, the mechanisms underlying perfusion defects in LNs during the early stage of LN metastasis have not been investigated. We show that tumor mass formation with very few microvessels was associated with a perfusion defect in a non-enlarged LN at the early stage of LN metastasis in a LN adenopathy mouse (LN size circa 10 mm). We found in a mouse model of LN metastasis, induced using non-keratinizing tumor cells, that during the formation of the perfusion defect in a non-enlarged LN, the number of blood vessels ≤ 50 µm in diameter decreased, while those of > 50 µm in diameter increased. The methods used were contrast-enhanced high-frequency ultrasound and contrast-enhanced micro-CT imaging systems, with a maximum spatial resolution of > 30 µm. Furthermore, we found no tumor angiogenesis or oxygen partial pressure (pO2) changes in the metastatic LN. Our results demonstrate that the perfusion defect appears to be a specific form of tumorigenesis in the LN, which is a vascular-rich organ. We anticipate that a perfusion defect on ultrasound, CT or MRI images will be used as an indicator of a non-enlarged metastatic LN at an early stage.

Study of the physicochemical properties of drugs suitable for administration using a lymphatic drug delivery system.

Lymph node (LN) metastasis is thought to account for 20-30% of deaths from head and neck cancer. The lymphatic drug delivery system (LDDS) is a new technology that enables the injection of drugs into a sentinel LN (SLN) during the early stage of tumor metastasis to treat the SLN and secondary metastatic LNs. However, the optimal physicochemical properties of the solvent used to carry the drug have not been determined. Here, we show that the osmotic pressure and viscosity of the solvent influenced the antitumor effect of cisplatin (CDDP) in a mouse model of LN metastasis. Tumor cells were inoculated into the proper axillary LN (PALN), and the LDDS was used to inject CDDP solution into the subiliac LN (SiLN) to treat the tumor cells in the downstream PALN. CDDP dissolved in saline had no therapeutic effects in the PALN after it was injected into the SiLN using the LDDS or into the tail vein (as a control). However, CDDP solution with an osmotic pressure of ~ 1,900 kPa and a viscosity of ~ 12 mPa⋅s suppressed tumor growth in the PALN after it was injected into the SiLN using the LDDS. The high osmotic pressure dilated the lymphatic vessels and sinuses to enhance drug flow in the PALN, and the high viscosity increased the retention of CDDP in the PALN. Our results demonstrate that optimizing the osmotic pressure and viscosity of the solvent can enhance the effects of CDDP, and possibly other anticancer drugs, after administration using the LDDS.

Treatment of false-negative metastatic lymph nodes by a lymphatic drug delivery system with 5-fluorouracil.

Metastatic lymph nodes (LNs) may be the origin of systemic metastases. It will be important to develop a strategy that prevents systemic metastasis by treating these LNs at an early stage. False-negative metastatic LNs, which are found during the early stage of metastasis development, are those that contain tumor cells but have a size and shape similar to LNs that do not host tumor cells. Here, we show that 5-fluorouracil (5-FU), delivered by means of a novel lymphatic drug delivery system (LDDS), can treat LNs with false-negative metastases in a mouse model. The effects of 5-FU on four cell lines were investigated using in vitro cytotoxicity and cell survival assays. The therapeutic effects of LDDS-administered 5-FU on false-negative metastatic LNs were evaluated using bioluminescence imaging, high-frequency ultrasound (US), and histology in MHX10/Mo-lpr/lpr mice. These experimental animals develop LNs that are similar in size to human LNs. We found that all cell lines showed sensitivity to 5-FU in the in vitro assays. Furthermore, a concentration-dependent effect of 5-FU to inhibit tumor growth was observed in tumor cells with low invasive growth characteristics, although a significant reduction in metastatic LN volume was not detected in MHX10/Mo-lpr/lpr mice. Adverse effects of 5-FU were not detected. 5-Fluorouracil administration with a LDDS is an effective treatment method for false-negative metastatic LNs. We anticipate that the delivery of anticancer drugs by a LDDS will be of great benefit in the prevention and treatment of cancer metastasis via LNs.