Intravital imaging of lymph node metastasis reveals its ability to contribute to distant metastasis
1Edwin L. Steele Laboratories, Department of Radiation Oncology, MGH Cancer Center, Massachusetts General Hospital and Harvard Medical School, Boston, United States
Background: The presence of lymph node metastasis in patients with solid tumors is associated with tumor aggressiveness, poorer prognosis and the recommendation for systemic therapy. However, whether tumor cells exit the lymph node and contribute to distant metastases remains controversial. Evidence shows that treating disease in the lymph node improves survival in some patients. In this study, we address the controversy of whether tumor cells colonizing the axillary lymph nodes could disseminate to distant sites.
Materials and Methods: We used syngeneic murine cell lines representing breast (4T1), melanoma (B16F10), head and neck (SCCVII) cancer that spontaneously metastasize to the lymph node. We engineered these cells to express Dendra2, a photoconvertable protein, to determine if metastatic tumor cells from the lymph node are able to seed distant organs like the lung. Using high-resolution multiphoton microscopy and a chronic lymph node window we monitored the behavior of tumor cells in the lymph node and their interaction with blood vessels.
Results: By photoconverting cancer cells only in lymph nodes, we show that spontaneous lymph node metastasis from breast cancer and melanoma mouse models can leave the lymph node and enter the systemic blood circulation. Our data show that the circulating tumor cells originating from the lymph node are viable and are able to proliferate in vitro. Using a combination of intravital microscopy and immunofluorescence staining, our data show that 7.4%±1.8% of isolated tumor cells in the lymph node are intraluminal and an additional 16.3%±1.9% were within 5μm of a blood vessel compared to an expected 8.8%±0.9% if they were randomly distributed. Further, we identified lung micrometastases that originated from the lymph node in both mouse models. Analysis of human metastatic lymph node samples from head and neck cancer patients revealed perivascular association of isolated tumor cells as well as the presence of cancer cells inside blood vessels similar to our observations in the mouse models.
Conclusions: Together, our data show for the first time that in spontaneous breast and melanoma mouse models, tumor cells in the lymph node can invade blood vessels, exit the lymph node and colonize distant organs like the lung. These studies exemplify the importance of treating metastatic disease in the lymph node, which could potentially lead to distant metastasis.
Keywords: Lymph node, metastasis, circulating tumor cells, intravital imaging, distant metastasis
Nanoparticle to treat established lymph node metastasis and primary tumor
1Department of Biomedical Engineering, University of Florida, Gainesville, United States
Background: Lymph node metastasis increases the probability of cancer relapse and spread to other tissues even after resection of the primary tumor. Here we demonstrate nanoparticles (NPs) that efficiently transport chemotherapy to lymph nodes with metastasis and primary tumor for effective cancer treatment.
Materials and Methods: Nanoparticles (NPs) were formulated from poly (glycolide-co-lactide) using a modified single emulsion-solvent evaporation technique. The NPs were characterized for size, charge and surface morphology. Biodistribution of NPs into lymph nodes and tumors was determined after intravenous (IV) and subcutaneous (SQ) injection in a murine model of prostate cancer. Efficacy of paclitaxel (PTX) loaded NPs at inhibiting the progression of both established tumor and lymph node metastases were also evaluated.
Results: The formulated NPs which were 112 nm in size localized into all lymph nodes along a targeted lymphatic network after SQ injection (Fig.1A). Lymph nodes with metastasis showed a 6-fold greater NP accumulation compared to normal lymph nodes. The NPs ultimately entered the blood and accumulated into tumors at 14% injected dose/g of tumor (ID/GT) compared to 1% ID/GT when injected IV. A single SQ injection of PTX-loaded NPs (PTX-NP-SQ) in murine prostate cancer models with established tumors and lymph node metastasis decreased metastasis burden by 500% (Fig.1B) and increased tumor doubling time by 40% compared to IV injection (PTX-NP-IV). This resulted in 20% increase in average survival time compared to those injected intravenously.
Conclusions: These results demonstrate that NPs can effectively deliver therapeutics primary tumors and lymph node metastases for better cancer treatment.
Cutaneous Immune Factors and Regression of In-Transit/Lymphatic Melanoma After Intralesional Injection of Bacille Calmette-Guerin
1Division of Surgical Oncology, John Wayne Cancer Institute, Providence Saint John’s Health Center, Santa Monica, CA, Santa Monica, United States
Mycobacterium bovis Bacille Calmette-Guerin (BCG) is a known intralesional (ILBCG) therapeutic option for stage III in-transit/lymphatic melanoma. The mechanism of action is unknown; however, regressions in up to 50% of injected lesions and 17% of uninjected lesions have been reported. BCG and other mycobacteria express ligands capable of stimulating γ9δ2 T cells. We hypothesize that γ9δ2 T cells play a role in promoting BCG-mediated antitumor immunity in patients treated with ILBCG.
Materials and Methods:
Eight patients were diagnosed with stage III in-transit/lymphatic melanoma and treated with ILBCG per study protocol. BCG injected and uninjected lesions were resected and subjected to immunohistochemistry and RNAseq gene expression analyses.
BCG injection induced a significant increase of γ9δ2 T cell infiltration in BCG injected lesions as determined by immunohistochemistry and RNAseq gene expression analyses. Furthermore, BCG injection elicited: the expression of chemokines (such as CXCL9, 10, and 11) capable of attracting γδT cells, the expression of antigenic ligands (BTN3A1) capable of activating γ9δ2 T cells in BCG injected lesions, and the secretion of cytokines by activated γδ T cells. Interestingly, we found that γδ T cell infiltration was associated with the regression of BCG uninjected lesions.
These data strongly suggest that γ9δ2 T cells contribute to in transit/lymphatic melanoma regressions induced by ILBCG. γ9δ2 T cells may serve as a useful target for future immunotherapeutic study.
Epigenetic alterations of spliceosome factors are associated with organ-specific melanoma metastasis
1Department of Translational Molecular Medicine, John Wayne Cancer Institute at Providence Saint John’s Health Center, Santa Monica, United States
Background: Metastatic melanoma cells exhibit an extraordinary phenotypic plasticity, not only in adapting to unfamiliar microenvironments, but also in surviving aggressive treatments and immune response. A major source of phenotypic variability is the alternative splicing of the messenger RNA (mRNA). This process is catalyzed by one of the most complex cellular machineries, the spliceosome, which is composed of ribonucleoproteins and polypeptides denominated spliceosome factors (SF). Changes in the expression level of SFs lead to a splicing reprogramming that has been linked to different types of cancer progression. We have recently demonstrated that the interplay between epigenomic and transcriptomic alterations of SFs generates melanoma cells with an enhanced predisposition for brain metastasis. Material and Methods: Using the Illumina Human 450K BeadChip technology, we generated genome-wide DNA methylation maps of 133 melanoma specimens, including melanocytes and nevi (n=9), primary melanomas (PRM; n=15), lymph node metastases (LNM; n=27), extracranial metastases (n=37), and brain metastases (MBM; n=45). This analysis included 15 patients with PRM or LNM paired with multiple (>2) distant organ metastases (DOM). To ensure highly representative DNA methylation patterns, all the specimens were microdissected. Additionally, DNA methylation and gene expression data for DOM (n=67) generated by the Cancer Genome Atlas (TCGA) were integrated to validate our findings. Results: This epigenomic profiling allowed us to evaluate changes in the DNA methylation levels of 6,370 genomic regions affecting 356 genes encoding splicing and mRNA editing factors. Overall, 58 genomic regions affecting 12 genes were differentially methylated during melanoma progression to metastasis (Absolute [PRM–DOM]>0.3; Wilcoxon test, FDR-p<0.01). In agreement with our previous studies, this analysis identified epigenetic alterations on ESRP1, ESRP2 and PTBP1 genes which, as we recently described, are involved in melanoma metastasis in an independent patient cohort. These regions were primarily located in gene body CpG island regions suggesting a positive influence on gene expression rates. Interestingly, liver metastases presented hypermethylation of 16 genomic regions overlapping the ADARB2 gene which regulates the splicing of pre-micro-RNAs. Conversely, bowel metastases showed hypomethylation of the ADARB2 gene. ESRP1 and ESRP2 genes were hypomethylated in spleen and liver metastases but hypermethylated in lung and brain metastases. Analysis of TCGA RNA-sequencing data showed changes in the splicing program of several targets of these SFs. Conclusions: Aberrant splicing has a significant impact on melanoma progression to metastasis. The characterization of epigenetic regulation of SF genes influenced by metastasis microenvironment opens up a new avenue for understanding the affinity of melanoma cells for specific metastatic niches.
Keywords: Epigenomic Regulation, Alternative Splicing, Melanoma Metastasis, DNA methylation
Innovations in Lymphatic Microsurgery – MLVA and FLA-LVSP: The Complete Treatment Package
Department of Plastic, Reconstructive & Aesthetic Surgery ,Unit for Lymphatic Microsurgery, Salus Hospital, GVM Care & Research, Reggio Emilia, Italy
Background: Despite the common assertion that little is understood about the pathophysiology of disorders of the lymphatic system and that satisfactory treatments are lacking, there is a rich history in medicine dating back close to 500 years of research on the lymphatic system. In the past 40 years there has been a burgeoning interest in research on, and clinical application for, diseases involving the lymphatic system. Development of microsurgical techniques has significantly advanced the field of Lymphology and the treatment of lymphedema secondary to oncological surgery.
Methods:The author reports on the development of microsurgery for lymphedema and new developments with particular reference to the wealth of experience in treating patients in Genoa, Italy. The “single-site” microsurgical technique is based on the identification, with the use of Patent Blue Dye / Fluorescent Micro-lymphography (PDE Test), of the lymhatics in the axillary or inguinal-crural regions (“single-site”) and the completion of derivative multiple lymphatic–venous anastomoses (MLVA). The lymphatics are anastomosed with telescopic technique to multiple tributary vein, for example, the axillary vein or the saphenous vein, depending on the affected limb.
In cases of advanced lymphedema, patients are treated by a recently developed Fibro-Lipo-Lymph-Aspiration technique to improve this chronic swelling, using a Lymph Vessel Sparing Procedure (FLLA-LVSP). Using microlymphography techniques to highlight the lymphatic pathways, the excess adipose tissue was carefully aspirated with the FLLA-LVSP procedure.
Results: With the “single-site” MLVA, 4000 patients obtained significant reductions in excess limb volume of over 84%, with an average follow-up of 15 years or more. Over 86% of patients with earlier stages of disease (stage IB or IIA) progressively stopped using conservative therapies and 42% of patients with later stages (stages IIB and III) decreased the frequency of physical therapies. DLA attacks considerably reduced by over 91%.
For 250 advanced cases, 0.80L on average for the upper limb and 2.42L for the lower limb was removed with the FLLA-LVSP. For the upper limb, there was an average pre-surgery excess volume of 20.19%, which reduced to 2.68% after the FLLA-LVSP (Z-score =-6.90, p<0.001). Similarly, for the lower limb, there was an average pre-surgery excess limb volume of 21.24% and a reduction to 2.64% post-operatively (Z-score=-3.57, p<0.01). No episodes of post-operative infection occurred. See: Figure 1, pre, post-MLVA, and post-FLLA-LVSP.
Conclusion: MLVA or techniques when performed at a single-site produce excellent outcomes in the treatment of secondary lymphedemas, giving the possibility of a complete restoration of lymphatic flow in early stages of disease when tissue changes are minimal. In cases of advanced lymphedema, the FLLA-LVSP is efficient. It is possible to complete an entire leg within 90 minutes. Recovery time is short and cosmetic results are immediate. More importantly, the removal of excess tissue is completed without further damage to lymphatic vessels, providing evidence of the efficacy of FLLA-LVSP in limb-reshaping, whilst maintaining the optimal lymphatic flow restored by Lymphatic Microsurgery. With this procedure, in some patients, it is possible to achieve almost 100% volume reduction and to eliminate the use of compressive stockings.
Keywords: “single-site” lymphatic microsurgery, advanced secondary lymphedema, liposuction, lymph vessel sparing, fibroadipose tissue,
Cryofluorescence Tomography for Single-Cell and Micron-Level of Analysis of Cancer Metastasis and the Lymphovascular System – Bridging the Microscopic/Mesoscopic Divide
1inviCRO, LLC, Boston, United States
Background: Fluorescence molecular imaging is a valuable imaging modality at both the cellular and the whole-body imaging regimes. Although valuable for pathological analysis, fluorescence histology has a severely limited field of view. Similarly, bulk fluorescence imaging (such as with epi-illumination schemes) is either highly surface weighted, or limited to a resolution of a few cubic millimeters at best using tomographic approaches. Cryofluorescence tomography (CFT) is proposed as an imaging scheme to bridge the gap between microscopic and mesoscopic imaging methods, while still exploiting the vast and growing library of fluorescent tracers.
CFT is comprised of serial cryoslicing and high resolution imaging of a tissue sample block at multiple visible and NIR wavelengths. For each slice, a series of fluorescence images is captured simultaneous with white light images. In this way, valuable molecular fluorescence information is gathered along with anatomical information. Furthermore, imaging is performed on the block face rather than on transferred slices, thus completely eliminating registration artifacts caused by tissue deformation, folding, and tearing. Imaging is performed by interfacing a cryoslicer (Leica 3050s) with a surgical fluorescence imaging system (FLARE model R1, Curadel, Marlborough, MA). This imaging scheme can be used to automatically recover 3D fluorescence molecular information at mesoscopic resolution, with imaging times of under an hour for a whole mouse.
Materials and Methods: To test this imaging scheme, a rat was injected intrathecally with a near-infrared fluorophore having peak emission at 800 nm and intravenously with a near-infrared fluorophore having peak emission at 700 nm. At one hour, the kidneys were resected and imaged using CFT with 25 micron slices to be able to follow tracer clearance from the CSF to the blood and blood to urine using near-infrared fluorescence. Basic structural analysis based on the white light images was also performed. A color-based segmentation was performed to segment the vasculature of the sample.
Results: A comparison of the different imaging channels (Figure) shows a snapshot comparison of the renal clearance of the two tracers through different injection routes at approximately 25 micron voxel size. The images show that it is possible to resolve molecular and structural features on the same order of magnitude as lymphatic channels and the vasculature.
Conclusions: Using this new CFT system, two independent biomarkers can be imaged simultaneously over an entire mouse or rat with resolution far superior to whole-body nuclear imaging methods. By utilizing FLARE® lipophilic cell tracking dyes, single cells can be labeled and tracked anywhere in the animal. This should be particularly useful for tracking the migration and homing of metastatic cells. Using NIR fluorescent targeting ligands, virtually any in vivo target can be assessed over large volumes of tissue. Multiple examples of CFT for imaging cancer metastasis, lymphovascular processes, and biomarkers will be presented.
Keywords: fluorescence, histology, cryoimaging, cryofluorescence