High-fat diet, but not duration of lactation, increases mammary gland lymphatic vessel function and subsequent growth of inflammatory breast cancer cells.

Inflammatory breast cancer (IBC) presents as rapid-onset swelling and breast skin changes caused by tumor emboli in the breast and breast skin lymphatics. IBC has been linked with obesity and duration of breastfeeding, but how these factors affect IBC tumor progression is not clear. We modeled the simultaneous effects of diet and weaning in mice on in vivo lymphatic function; on IBC tumor growth; and on aspects of the mammary gland microenvironment before and after IBC (SUM149) xenograft inoculation. We hypothesized that weaning status and diet would have synergistic effects on lymphatic function and the breast microenvironment to enhance IBC tumor growth. Changes in lymphatic structure and function were characterized with in vivo near-infrared fluorescence (NIRF) imaging. Mice were fed either a high-fat diet (HFD; 60 kcal%) or a normal/low-fat diet (LFD; 10 kcal%), bred twice, and subjected to either normal-duration nursing (NW) or forced weaning (FW). SUM149 IBC tumors were implanted at 14 months; images were obtained before and after implantation. Multiparous mice fed HFD showed increased pre-tumor lymphatic pulsing in both the FW and NW groups relative to mice fed LFD. HFD promoted tumor growth independent of weaning time (P = 0.04). Pre-tumor lymphatic pulsing was associated with tumor volume at 8 weeks (P = 0.02) and was significantly correlated with expression of the lymphatic tracking ligand CCL21 (P = 0.05, Table 1). HFD significantly increased the numbers of monocyte-derived IBA1+, CD163+, and CD11c+ cells (P < 0.0001, P < 0.0001, P = 0.0005) in the contralateral, non-tumor-bearing mammary gland. Numbers of lymphangiogenic podoplanin+/IBA1+ macrophages were increased in the ducts of HFD and FW mice (all P < 0.003). HFD in nulliparous mice had a similar increase in lymphatic pulsing at 14 weeks (P = 0.006), indicating that this functional change was independent of parity. We conclude that HFD induced increases in mammary gland lymphatic function, assessed as pulsing rate before tumor initiation, and correlated with inflammation in the mammary gland and increased SUM149 tumor growth. The relationship between diet, lymphatic pulsing, and tumor growth warrants further investigation.

Nanotopography-based lymphatic delivery for improved anti-tumor responses to checkpoint blockade immunotherapy.

Rationale: Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is a co-inhibitory checkpoint receptor that is expressed by naïve T-cells in lymph nodes (LNs) to inhibit activation against "self" antigens (Ags). In cancer, anti-CTLA-4 blocks inhibitory action, enabling robust activation of T-cells against tumor Ags presented in tumor draining LNs (TDLNs). However, anti-CTLA-4 is administered intravenously with limited exposure within TDLNs and immune related adverse events (irAEs) are associated with over-stimulation of the immune system. Methods: Herein, we first deliver anti-CTLA-4 in an orthotopic mammary carcinoma murine model using a nanotopographical microneedle-array device to compare its anti-tumor response to that from systemic administration. Additionally, to demonstrate the feasibility of lymphatic delivery in humans using the device, we use near-infrared fluorescence imaging to image delivery of ICG to LNs. Results: Our data show that lymphatic infusion results in more effective tumor growth inhibition, arrest of metastases, increased tumor infiltrating lymphocytes and complete responses when compared to conventional systemic administration. In clinical studies, we demonstrate for the first time that nanotopographic infusion can deliver ICG through the lymphatics directly to the axilla and inguinal LNs of healthy human volunteers. Conclusion: Taken together, these results suggest that regional delivery using a nanotopography-based microneedle array could revolutionize checkpoint blockade immunotherapy by reducing systemic drug exposure and maximizing drug delivery to TDLNs where tumor Ags present. Future work is needed to determine whether lymphatic delivery of anti-CTLA-4 can alleviate irAEs that occur with systemic dosing.

Radiation Dose-Dependent Changes in Lymphatic Remodeling.

PURPOSE: Postoperative radiation therapy (RT) delivered to lymphatics is associated with an increased risk of developing lymphedema. Reported effects of RT on lymphatic vessels have varied, however, possibly because of the use of different animal models with varying surgery and radiation schedules and the inability to directly and longitudinally image lymphatics in vivo. Here we report, using noninvasive imaging, changes in lymphatic remodeling and function in response to surgery and RT in a mouse model. METHODS AND MATERIALS: Popliteal lymphadenectomy in mice preceded single-dose gamma irradiation of the lower extremity at a single dose of 0, 20, or 40 Gy. The right hind limb of intact mice was also radiated with 4 fractions (4 × 5 Gy). Near-infrared fluorescence lymphatic imaging with indocyanine green was performed over 6 months to monitor lymphatic vessel remodeling. RESULTS: Postoperative mice treated with 20 Gy showed transient changes in lymphatic drainage, exacerbated vessel remodeling including qualitative vessel dilation and abnormal indocyanine green pooling from week 1 to 2, and initiation of restoration of lymphatic vessels, although dermal backflow was occasionally observed. Mice treated with 40 Gy showed steadily increasing lymphatic impairment until week 3 and extravasation of dye and dermal backflow in weeks 4 to 25. The ankles of mice treated with 40 Gy were significantly swollen from weeks 2 to 4 as compared with mice treated with 0 Gy or 20 Gy. Mice that received fractionated RT exhibited lymphatic vessel remodeling similar to remodeling that occurred when a single 20 Gy dose was given; however, dermal backflow did not resolve as it did in the case of a single 20 Gy dose. CONCLUSIONS: The degree of nonreversing lymphatic damage seen in our mouse model was dependent on RT dose. Our results suggest that near-infrared fluorescence lymphatic imaging detection of early lymphatic changes can be used to predict development of lymphedema in patients with cancer.

Impaired Peripheral Lymphatic Function and Cerebrospinal Fluid Outflow in a Mouse Model of Alzheimer's Disease.

Cerebrospinal fluid (CSF) outflow from the brain occurs through absorption into the arachnoid villi and, more predominantly, through meningeal and olfactory lymphatics that ultimately drain into the peripheral lymphatics. Impaired CSF outflow has been postulated as a contributing mechanism in Alzheimer's disease (AD). Herein we conducted near-infrared fluorescence imaging of CSF outflow into the peripheral lymph nodes (LNs) and of peripheral lymphatic function in a transgenic mouse model of AD (5XFAD) and wild-type (WT) littermates. CSF outflow was assessed from change in fluorescence intensity in the submandibular LNs as a function of time following bolus, an intrathecal injection of indocyanine green (ICG). Peripheral lymphatic function was measured by assessing lymphangion contractile function in lymphatics draining into the popliteal LN following intradermal ICG injection in the dorsal aspect of the hind paw. The results show 1) significantly impaired CSF outflow into the submandibular LNs of 5XFAD mice and 2) reduced contractile frequency in the peripheral lymphatics as compared to WT mice. Impaired CSF clearance was also evidenced by reduction of fluorescence on ventral surfaces of extracted brains of 5XFAD mice at euthanasia. These results support the hypothesis that lymphatic congestion caused by reduced peripheral lymphatic function could limit CSF outflow and may contribute to the cause and/or progression of AD.

Near-infrared fluorescence lymphatic imaging in vascular endothelial growth factor-C overexpressing murine melanoma.

In this study we employ a near-infrared fluorescence lymphatic imaging (NIRFLI) technique to longitudinally image spatial and temporal changes in the lymphatics in mice bearing vascular endothelial growth factor (VEGF)-C overexpressing B16F10 (VEGF-C-B16F10) or mock-transduced B16F10 (mock-B16F10) melanoma tumors. Our NIRFLI data show that ICG-laden lymph accumulates into a VEGF-C-B16F10 tumor compared to mock-B16F10 at 3 days post implantation, presumably due to increased lymphatic vessel permeability. Quantification shows a significantly greater percentage of ICG-perfused area in VEGF-C-B16F10 (7.6 ± 2) as compared to MOCK-B16F10 (1 ± 0.5; p = 0.02), which is also confirmed by quantification of the lymphatic leakage of evans blue dye (optical density at 610nm; VEGF-C-B16F10, 10.5 ± 2; mock-B16F10, 5.1 ± 0.5; p = 0.009); thereafter, lymphatic leakage is visualized only in the peritumoral region. Our imaging data also show that anti-VEGF-C treatment in VEGF-C-B16F10 restores normal lymphatic vessel integrity and reduces dye extravasation. Because NIRFLI technology can be used to non-invasively detect lymphatic changes associated with cancer, it may provide a new diagnostic to assess the lack of lymphatic vessel integrity that promotes lymphovascular invasion and to assess therapies that could arrest invasion through normalization of the lymphatic vasculature.

Fluorescence imaging of lymphatic outflow of cerebrospinal fluid in mice.

Cerebrospinal fluid (CSF) is known to be reabsorbed by the lymphatic vessels and drain into the lymph nodes (LNs) through peripheral lymphatic vessels. In the peripheral lymphatics, the contractile pumping action of lymphangions mediates lymph drainage; yet it is unknown whether lymphatic vessels draining cranial and spinal CSF show similar function. Herein, we used non-invasive near-infrared fluorescence imaging (NIRFI) to image (i) indocyanine green (ICG) distribution along the neuraxis and (ii) routes of ICG-laden CSF outflow into the lymphatics following intrathecal lumbar administration. We demonstrate lymphatic contractile function in peripheral lymphatics draining from the nasal lymphatics to the mandibular LNs. In addition, we observed afferent sciatic lymphatic vessels, which also show contractile activity and transport spinal CSF into the sciatic LNs. This drainage pattern was also visualized by NIRFI following intrathecal thoracic injection. In situ intravital imaging following intrathecal lumbar injection of blue dye shows similar distributions to that seen in vivo with ICG. NIRFI could be used as a tool to probe CSF pathology including neurological disorders by imaging CSF outflow dynamics to lymphatics.