Docetaxel administered through a novel lymphatic drug delivery system (LDDS) improved treatment outcomes for lymph node metastasis.

Recently, sentinel lymph nodes (LNs) have been recognized as a starting point of hematogenous metastasis; thus, an increase in the control rate of LN metastasis is expected to improve the survival rate. Although surgical treatment and radiation therapy are commonly used for the radical treatment of LNs, these treatments are associated with lymphedema, pain, and an extended hospital stay. In a recent mouse study, activation of metastatic tumors in distant organs was reported after removing LNs, with or without metastasis to the LNs. Thus, there is the necessity for cancer treatment that can replace LN removal. Here, we evaluated the treatment efficacy of lymphatic drug delivery system (LDDS) with osmotic pressure and viscosity escalated Docetaxel at the early stage of LN metastasis. MXH10/Mo/lpr mice were inoculated with mouse breast cancer cells into Subiliac LN to create the metastatic mouse model. Docetaxel was injected into mouse mammary carcinoma cells inoculated LN as a single shot (SS) or double shot (DS) to understand the therapeutic mechanism of a single shot or double shot intervention using an in vivo imaging system, histology, and qPCR. The results showed that the DS administration of docetaxel at 1,960 kPa (12 mPa∙s) had better therapeutic outcomes with increased complete response and improved survival with reduced adverse events. The results also revealed that administration of a DS of docetaxel enhances differentiation of T helper cells, and improves survival and therapeutic outcomes. From a safety perspective, LDDS-administered DS of low-concentration docetaxel without any other anticancer treatments to LNs a novel approach to cancer management of LN metastasis. We emphasize that LDDS is a groundbreaking method of delivering anticancer drugs specifically to cancer susceptible LNs and is designed to enhance the effectiveness of cancer treatment while minimizing side effects.

Intralymphatic injection of chemotherapy drugs modulated with glucose improves their anticancer effect.

Lymph node metastasis (LNM) has a significant impact on cancer prognosis, emphasizing the need for effective treatment strategies. This study investigated the potential use of high osmotic pressure drug solutions with low viscosity administration using a lymphatic drug delivery system (LDDS) to improve LNM treatment outcomes. The hypothesis was that injection of epirubicin or nimustine at high osmotic pressure but without altered viscosity would enhance drug retention and accumulation in LNs, thereby improving the efficacy of treatment. Biofluorescence analysis revealed enhanced drug accumulation and retention in LNs after administration using LDDS compared to intravenous (i.v) injection. Histopathological results demonstrated minimal tissue damage in the LDDS groups. Pharmacokinetic analysis revealed an improved treatment response with higher drug accumulation and retention in LNs. The LDDS approach offers the potential for greatly reduced side effects of chemotherapy drugs, lower dosage requirements and crucially increased drug retention in LNs. The results highlight the promise of high osmotic pressure drug solutions with low viscosity administrated using the LDDS for enhancing the treatment efficacy of LN metastasis. Further research and clinical trials are warranted to validate these results and optimize the clinical translation of this novel treatment technique.

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.

Combination therapy of lymphatic drug delivery and total body irradiation in a metastatic lymph node and lung mouse model.

Chemotherapy using a lymphatic drug delivery system (LDDS) targeting lymph nodes (LNs) in the early stage of metastasis has a superior antitumor effect to systemic chemotherapy. An LDDS produces a higher drug retention rate and tissue selectivity in LNs. To expand the therapeutic coverage of LDDS from local treatment of metastatic LNs to prevention of distant metastases, the combination of treatment with therapies that enhance systemic tumor immune effects is an important therapeutic strategy. Recently, total body irradiation (TBI) has been shown to activate immune responses and alter the tumor microenvironment. Here we show that combination therapy with TBI and LDDS improves the antitumor effect of metastatic LNs and lung metastasis. Tumor cells were inoculated into the subiliac LN (SiLN) to induce metastasis into the proper axillary LN (PALN) and lung in a mouse model. TBI was carried out on day 4 after inoculation using a gamma irradiator. Lymphatic drug delivery into the accessory axillary LN was used to treat PALN. In vivo bioluminescence imaging, high-frequency ultrasound, and histology showed that combination therapy using TBI (total dose 1.0 Gy once) and the LDDS suppressed tumor growth in LNs and lung metastases and was more effective than using LDDS or TBI alone. Quantitative RT-PCR of spleens after combination therapy revealed increased expression of CD4, CD8, and IL-12b, indicating an activated immune response. The results show that combination therapy with TBI and LDDS is a method to improve the efficacy of LN metastases and distant metastases therapy and is a promising novel approach to treat cancer patients.

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.

Protocols for the Evaluation of a Lymphatic Drug Delivery System Combined with Bioluminescence to Treat Metastatic Lymph Nodes.

Bioluminescence (BL) imaging is a powerful non-invasive imaging modality widely used in a broad range of biological disciplines for many types of measurements. The applications of BL imaging in biomedicine are diverse, including tracking bacterial progression, research on gene expression patterns, monitoring tumor cell growth/regression or treatment responses, determining the location and proliferation of stem cells, and so on. It is particularly valuable when studying tissues at depths of 1 to 2 cm in mouse models during preclinical research. Here we describe the protocols for the therapeutic evaluation of a lymphatic drug delivery system (LDDS) using an in vivo BL imaging system (IVIS) for the treatment of metastatic lymph nodes (LNs) with 5-fluorouracil (5-FU). The LDDS is a method that directly injects anticancer drugs into sentinel LNs (SLNs) and delivers them to their downstream LNs. In the protocol, we show that metastases in the proper axillary LN (PALN) are induced by the injection of luciferase-expressing tumor cells into the subiliac LN (SiLN) of MXH10/Mo-lpr/lpr (MXH10/Mo/lpr) mice. 5-FU is injected using the LDDS into the accessory axillary LN (AALN) to treat tumor cells in the PALN after the tumor cell growth is confirmed in the PALN. The tumor growth and therapeutic effects are evaluated by IVIS. This method can be used to evaluate tumor growth and efficacy of anticancer drugs/particles, radiotherapy, surgery, and/or a combination of these methods in various experimental procedures in the oncology field.

Intranodal delivery of modified docetaxel: Innovative therapeutic method to inhibit tumor cell growth in lymph nodes.

Delivery of chemotherapeutic agents into metastatic lymph nodes (LNs) is challenging as they are unevenly distributed in the body. They are difficult to access via traditional systemic routes of drug administration, which produce significant adverse effects and result in low accumulation of drugs into the cancerous LN. To improve the survival rate of patients with LN metastasis, a lymphatic drug delivery system (LDDS) has been developed to target metastatic LN by delivering chemotherapy agents into sentinel LN (SLN) under ultrasound guidance. The LDDS is an advanced method that can be applied in the early stage of the progression of tumor cells in the SLN before tumor mass formation has occurred. Here we investigated the optimal physicochemical ranges of chemotherapeutic agents' solvents with the aim of increasing treatment efficacy using the LDDS. We found that an appropriate osmotic pressure range for drug administration was 700-3,000 kPa, with a viscosity < 40 mPa⋅s. In these physicochemical ranges, expansion of lymphatic vessels and sinuses, drug retention, and subsequent antitumor effects could be more precisely controlled. Furthermore, the antitumor effects depended on the tumor progression stage in the SLN, the injection rate, and the volumes of administered drugs. We anticipate these optimal ranges to be a starting point for developing more effective drug regimens to treat metastatic LN with the LDDS.

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.

Intranodal pressure of a metastatic lymph node reflects the response to lymphatic drug delivery system.

Cancer metastasis to lymph nodes (LNs) almost certainly contributes to distant metastasis. Elevation of LN internal pressure (intranodal pressure, INP) during tumor proliferation is associated with a poor prognosis for patients. We have previously reported that a lymphatic drug delivery system (LDDS) allows the direct delivery of anticancer drugs into the lymphatic system and is a promising treatment strategy for early-stage LN metastasis. However, methods for evaluating the treatment effects have not been established. Here, we used a mouse model of MXH10/Mo-lpr/lpr, which develops a systemic swelling of LNs, and murine malignant fibrous histiocytoma-like (KM-Luc/GFP) cells or murine breast cancer (FM3A-Luc) cells inoculated into the subiliac LN of mice to produce a tumor-bearing LN model. The changes in INP during intranodal tumor progression and after treatment with cis-dichlorodiammineplatinum(II) (CDDP) using an LDDS were measured. We found that tumor progression was associated with an increase in INP that occurred independently of LN volume changes. The elevation in INP was suppressed by CDDP treatment with the LDDS when intranodal tumor progression was significantly inhibited. These findings indicate that INP is a useful parameter for monitoring the therapeutic effect in patients with LN metastasis who have been given drugs using an LDDS, which will serve to manage cancer metastasis treatment and contribute to an improved quality of life for cancer patients.

A model system for studying superselective radiotherapy of lymph node metastasis in mice with swollen lymph nodes.

Utilizing mice with swollen lymph nodes, we succeeded in irradiating individual metastatic lymph nodes through a hole in a lead shield. This system enabled us to increase the radiation dose to >8 Gy (the lethal dose for total-body irradiation) and evaluate both direct and abscopal antitumor effects.

Analysis of tumor vascularization in a mouse model of metastatic lung cancer.

Therapies targeting tumor vasculature would improve the treatment of lung metastasis, although the early changes in vascular structure are incompletely understood. Here, we show that obstructive metastatic foci in lung arterioles decrease the pulmonary vascular network. To generate a mouse model of lung metastasis activation, luciferase-expressing tumor cells were inoculated into the subiliac lymph node (SiLN) of an MXH10/Mo-lpr/lpr mouse, and metastatic tumor cells in the lungs were activated by SiLN resection. Activation of metastases was monitored by in vivo bioluminescence imaging. Pulmonary blood vessel characteristics were analyzed using ex vivo micro-computed tomography. The enhanced permeability and retention (EPR) effect in neovasculature after tumor cell activation was evaluated from the accumulation of intravenously injected indocyanine green (ICG) liposomes. Metastatic foci in lung arterioles were investigated histologically. Micro-computed tomography revealed decreases in pulmonary blood vessel length, volume and number of branching nodes during the early stage of metastasis caused by metastatic foci. ICG liposome accumulation by the EPR effect was not detected. Histology identified metastatic foci in lung arterioles. The lack of an EPR effect after the formation of metastatic foci in lung arterioles makes conventional systemic chemotherapy ineffective for lung metastasis. Thus, alternative therapeutic methods of drug delivery are needed.

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.

Lymph node resection induces the activation of tumor cells in the lungs.

Lymph node (LN) dissection is a crucial procedure for cancer staging, diagnosis and treatment, and for predicting patient survival. Activation of lung metastatic lesions after LN dissection has been described for head and neck cancer and breast cancer. Preclinical studies have reported that dissection of a tumor-bearing LN is involved in the activation and rapid growth of latent tumor metastases in distant organs, but it is also important to understand how normal (non-tumor-bearing) LN resection influences secondary cancer formation. Here, we describe how the resection of tumor-bearing and non-tumor-bearing LN affects distant metastases in MXH10/Mo-lpr/lpr mice. Tumor cells were administered intravenously and/or intranodally into the right subiliac lymph node (SiLN) to create a mouse model of lung metastasis. Luciferase imaging revealed that tumor cells in the lung were activated after resection of the SiLN, irrespective of whether it contained tumor cells. No luciferase activity was detected in the lungs of mice that did not undergo LN resection (excluding the intravenous inoculation group). Our results indicate that resection of an LN can activate distant metastases regardless of whether the LN contains tumor cells. Hence, lung metastatic lesions are suppressed while metastatic LN are present but activated after LN resection. If this phenomenon occurs in patients with cancer, it is likely that lung metastatic lesions may be activated by elective LN dissection in clinical N0 cases. The development of minimally invasive cancer therapy without surgery would help to minimize the risk of activation of distant metastatic lesions by LN resection.

Superselective Drug Delivery Using Doxorubicin-Encapsulated Liposomes and Ultrasound in a Mouse Model of Lung Metastasis Activation.

Conventional treatment of lymph node metastasis involves dissection of the tumor and regional lymph nodes, but this may cause activation of latent metastatic tumor cells. However, there are few reports on animal models regarding the activation of latent metastatic tumor cells and effective methods of treating activated tumor cells. Here, we report the use of a superselective drug delivery system in a mouse model of lung metastasis in which activated tumor cells are treated with doxorubicin-encapsulated liposomes (DOX-LP) and ultrasound. The axillary lymph node was injected with DOX-LP and exposed to ultrasound so that the released DOX would be delivered from the axillary lymph node to the metastatic lung via the subclavian vein, heart and pulmonary artery. The size of the DOX-LP was optimized to a diameter of 460 nm using indocyanine green-encapsulated liposomes, and the ultrasound intensity was 0.5 W/cm2. We found that compared with DOX or DOX-LP alone, the superselective drug delivery system was effective in the treatment of metastasis in both the lung and axillary lymph node. We anticipate that this superselective drug delivery system will be a starting point for the development of new techniques for treating lung metastasis in the clinical setting. Furthermore, the superselective drug delivery system may be used to screen novel drugs for the treatment of lung cancer and investigate the mechanisms of tumor cell activation after resection of a primary tumor or lymph nodes.

A novel treatment for metastatic lymph nodes using lymphatic delivery and photothermal therapy.

Systemic delivery of an anti-cancer agent often leads to only a small fraction of the administered dose accumulating in target sites. Delivering anti-cancer agents through the lymphatic network can achieve more efficient drug delivery for the treatment of lymph node metastasis. We show for the first time that polymeric gold nanorods (PAuNRs) can be delivered efficiently from an accessory axillary lymph node to a tumor-containing proper axillary lymph node, enabling effective treatment of lymph node metastasis. In a mouse model of metastasis, lymphatic spread of tumor was inhibited by lymphatic-delivered PAuNRs and near-infrared laser irradiation, with the skin temperature controlled by cooling. Unlike intravenous injection, lymphatic injection delivered PAuNRs at a high concentration within a short period. The results show that lymphatic administration has the potential to deliver anti-cancer agents to metastatic lymph nodes for inhibition of tumor growth and could be developed into a new therapeutic method.

Evaluation of the enhanced permeability and retention effect in the early stages of lymph node metastasis.

Most solid cancers spread to new sites via the lymphatics before hematogenous dissemination. However, only a small fraction of an intravenously administered anti-cancer drug enters the lymphatic system to reach metastatic lymph nodes (LN). Here, we show that the enhanced permeability and retention (EPR) effect is not induced during the early stages of LN metastasis. Luciferase-expressing tumor cells were injected into the subiliac LN of the MXH10/Mo-lpr/lpr mouse to induce metastasis to the proper axillary LN (PALN). In vivo biofluorescence imaging was used to confirm metastasis induction and to quantify the EPR effect, measured as PALN accumulation of intravenously injected indocyanine green (ICG) liposomes. PALN blood vessel volume changes were measured by contrast-enhanced high-frequency ultrasound imaging. The volume and density of blood vessels in the PALN increased until day 29 after inoculation, whereas the LN volume remained constant. ICG retention was first detected on day 29 post-inoculation. While CD31-positive cells increased up to day 29 post-inoculation, α-smooth muscle actin-positive cells were detected on day 29 post-inoculation for the first time. These results suggest that the EPR effect was not induced in the early stages of LN metastasis; therefore, systemic chemotherapy would likely not be beneficial during the early stages of LN metastasis. The development of an alternative drug delivery system, independent of the EPR effect, is required for the treatment of LN metastasis.