Rewiring the Lymphatic Landscape: Disorders, Remodeling, and Cancer Progression

Abstract

The lymphatic system is essential for maintaining the body’s fluid balance, lipid absorption, and immune regulation. The dysfunction of the lymphatic system is associated with a wide spectrum of disorders. These disorders include primary and secondary lymphedema, congenital malformations, and lymphatic neoplasms. In cancer patients, lymphatic remodeling is essential, which facilitates tumor progression and metastasis, while tertiary lymphoid structures (TLSs) develop during chronic inflammation and may be involved in anti-tumor immunity. This review highlights the immunological basis of lymphatic disorders, with a particular focus on cellular and molecular biomarkers that define disease states. The recent advances in molecular imaging techniques, such as ultrasonography (US), computed tomography (CT), and magnetic resonance lymphography (MRL), have improved and identified the diagnosis and therapeutic targets for lymphedema. Moreover, nanobiotechnology and nano-delivery tools have further enhanced the visibility of cancer cells by imaging. Artificial Intelligence (AI) in lymphatic systems have offered a new spectrum for disease prediction using forms of AI such as natural language processing (NLP), machine learning (ML), robotics-assisted approaches, fussy model (FM), and natural language processing (NLP)-based algorithms. Collectively, these advanced tools have improved diagnostic approaches and reveal exciting opportunities for future research and new therapeutic developments in patient care.

1. Lymphatic System

The lymphatic system is a highly organized network of vessels and lymphatic nodes important for immune cell transport, activation, and antigen presentation [1,2]. In addition to immunological function, the lymphatic system is essential for draining excess extracellular fluid from the body to the circulatory system [3,4]. It is also considered our body’s ‘sewerage system’. Based on functional characteristics, lymphoid organs are mainly classified into two categories: primary and secondary. Bone marrow and the thymus are primary lymphoid organs, where the primary repertoire of lymphocyte effector cells is generated. Secondary lymphoid organs include the spleen, tonsils, lymph nodes, and various mucosal tissues, which serve as reservoirs and sites of immune cell function [5].

The primary function of the lymphatic system is to maintain interstitial fluid balance by returning excess fluid derived from blood capillary filtrate and tissue immunosurveillance back into the bloodstream [6]. It also facilitates the transport of dietary fat and fat-soluble vitamins from the digestive tract into the venous circulation [4,6]. Once the pathogens invade our body’s first line of defense, the lymphatic system activates the adaptive immune response by trafficking lymphocytes. In the absence or dysfunction of the functional lymphatic system, the person may suffer from chronic edema and impaired immune responses, some of which can be serious and even life-threatening [3]. These clinical consequences underscore the need to better understand the lymphatic system in both health and disease. This review will provide a comprehensive overview of the lymphatic system and its role in homeostasis, major disorders with a particular focus on cancer, lymphangiogenesis, associated biomarkers, remodeling, and lymphatic neoplasms. Moreover, it also highlights therapeutic advances, the emerging applications of AI, and future directions for research and clinical applications.

1.1. Lymphatic Disorders

1.1.1. Lymphedema: A Major Concern

Lymphedema is one of the most common and recognizable lymphatic disorders. It is a chronic and progressive condition that results from the accumulation of lymph fluid, most often causing swelling in the lower limb. During this persistent inflammation, various immune cells, including CD4+ T cells and specialized M2-like macrophages, secreteseveral growth factors and cytokines, including TGF-β and IL-13, which are involved in tissue remodeling and contribute to disease progression [7,8].

1.1.1.1. Primary Lymphedema

This form of lymphedema is relatively rare and is linked to inherited genetic mutations affecting the development of lymphatic vessels [9,10]. Mutations in genes such as FLT4 (VEGFR-3), FOXC2, and GJC2 interfere with normal lymphatic growth and maintenance [9]. Clinically, primary lymphedema may be present at birth (Milroy disease) but can also arise during puberty (Meige disease) or later in adulthood [9].

1.1.1.2. Secondary Lymphedema

Secondary lymphedema is the most common form of lymphedema, which results from external damage to the lymphatic system [11]. In many low- and middle-income countries, it is most often caused by lymphatic filariasis, a parasitic nematode called Wuchereria bancrofti, which leads to permanent limb swelling [12]. In higher-income countries, cancer treatments like lymph node removal and radiation therapy (after breast cancer surgery, for instance) are the main causes of secondary lymphedema. In such patients, swelling may develop shortly after treatment or even several years later.

1.1.2. Congenital and Developmental Lymphatic Anomalies

Some lymphatic disorders arise from poorly developed lymphatics before birth. These are classified as congenital and developmental lymphatic anomalies, which include lymphatic malformations (LMs) and a group called complex lymphatic anomalies (CLAs). CLA includes Central Conducting Lymphatic Anomaly (CCLA), Generalized Lymphatic Anomaly (GLA), and Kaposiform Lymphangiomatosis (KLA) [9,13]. These conditions are characterized by abnormal vessel formation and disorganized lymphatic architecture, which lead to life-threatening complications. Understanding their underlying mechanisms is essential for diagnosis and the development of effective therapies.

1.1.3. Lymphatic Malformations (LMs)

LMs are benign but often problematic masses that typically occur around the head and neck region. LMs consist of clusters of abnormally formed lymphatic vessels, which are soft and sponge-like. Based on the size of the fluid-filled spaces, they are categorized as microcystic, macrocystic, or mixed. In this condition, somatic PIK3CA mutations are involved, which lead to abnormal growth signaling and cystic changes in lymphatic vessels [13,14]. These features make LMs clinically important as they can lead to significant functional complications.

1.1.4. Central Conducting Lymphatic Anomaly (CCLA)

CCLA involves abnormalities in the main lymphatic channels, such as the thoracic duct, which helps transport lymph fluid back into the body circulation. Malfunctioning of these channels causes disturbances in the lymph flow and leakage. When lymph flow is disrupted, fluid leaks into body cavities causing fluid build up around the lungs (chylothorax), in the abdomen (chylous ascites), and around the heart (chylopericardium). These disorders are collectively called chylous disorders [15].

1.1.5. Generalized Lymphatic Anomaly (GLA)

GLA, also referred to as lymphangiomatosis, is a rare disorder marked by the widespread growth of abnormal lymphatic vessels, which affect bones, lungs, spleen, and soft tissues. Mutations in PIK3CA and NRAS are involved in vessel growth [13,16]. During this disorder, tissues may show increased macrophage infiltration and high VEGF-C expression. Medications like sirolimus, which target mTOR pathways, are shown to have a positive correlation with improved disease outcomes [17,18].

1.1.6. Kaposiform Lymphangiomatosis (KLA)

KLA is a particularly aggressive type of lymphatic anomaly that shares common features with GLA but is characterized by distinctive spindle-shaped (kaposiform) cells. It involves severe clotting disturbances, such as elevated D-dimer and low fibrinogen [19,20].

1.1.7. Lymphatic Neoplasm

Lymphatic neoplasms are a group of cancers originating in the lymphatic system, commonly referred to as lymphomas, and broadly classified into Hodgkin lymphomas (HLs, 10%) and non-Hodgkin lymphomas (NHLs, ~90%) [21]. HL is defined by the presence of Reed–Sternberg (RS) cells, which can be further classified into classical (cHL) and nodular lymphocyte-predominant Hodgkin lymphoma (NLP-HL) [22,23]. In contrast, NHLs are categorized into two groups based on disease prognosis: indolent or aggressive types [24]. As the World Health Organization (WHO) classification has been consistently revised, most recently in 2022, the major groups and their histopathophysiology have been extensively reviewed [25,26]. Here, we provide a brief overview of the current lymphoma classifications.

1.1.7.1. Hodgkin Lymphoma

This group of lymphatic neoplasms is defined by the presence of characteristic neoplastic RS cells. These cells are large, multinucleated, and display two mirror-image nuclei within a reactive cellular background [23]. These cells are pathognomonic for cHL and are derived from germinal center B cells [27,28]. RS cells are typically negative for CD20 and CD45, which are only positive in neoplastic NLP-HL, but they are positive for CD15 and CD30 [23]. In addition, RS cells commonly express CD25, CD40, CD86, CD95 (Apo-1/Fas), HLA-DR, ICAM-1, PAX5, Fascin, and TRAF1. In contrast, NLP-HL lack typical RS cells but have lymphocytic cells, characterized by larger cells with folded, multilobulated nuclei.

1.1.7.2. Non-Hodgkin Lymphoma

Non-Hodgkin lymphomas (NHLs) are a diverse group of neoplasms that originate commonly from B cells which contribute to more than 80% of cases but less common from T cells and NK cells [29]. Mostly, NHL arises from chromosomal translocation, mutation, and different chromosomal abnormalities associated with different types of lymphoma [24]. Immunophenotypic analysis of NHL commonly includes B-cell-associated antigens (CD19, CD20, CD22, CD79a) and germinal center-associated markers (CD10 and BCL6), CD5, CD21, and FMC7 [24].

1.2. Infectious Lymphatic Disorders

Infections can also affect the lymphatic system, leading to conditions that require urgent attention.

1.2.1. Lymphangitis

This condition occurs when bacteria, most commonly Streptococcus pyogenes, enter the lymphatic vessels through a skin wound or infection. It typically presents with red, tender streaks that spread from the infection site towards nearby lymph nodes. This bacterial infection triggers a strong inflammatory response, including the activation of neutrophils and the production of several pro-inflammatory cytokines, such as IL-6 and TNF-α [30]. These conditions are often accompanied by fever and chills. If left untreated, lymphangitis can progress rapidly and lead to systemic infection, requiring prompt medical intervention.

1.2.2. Lymphadenitis

Lymphadenitis refers to swollen and inflamed lymph nodes from infections caused by bacteria, viruses, and fungi. It activates the germinal center and forms granuloma, similar to tuberculosis (TB) [31]. It may affect a particular group of nodes (localized lymphadenitis) or affect multiple regions of the body (generalized lymphadenitis). Because it is often a marker of underlying infection, lymphadenitis acts as an important clinical sign that needs further diagnostic evaluation.

1.2.3. Lymphatic Filariasis

Lymphatic filariasis, also known as elephantiasis, is caused by parasitic worms like Wuchereria bancrofti, which are transmitted through mosquito bites. The immune response in filariasis is predominantly mediated by Th2 cytokine production, which involves increased levels of IL-4, IL-5, and IgE production [32]. Although the infection is often acquired during childhood, the severe swelling of limbs or genitals usually appear later in life and leads to significant disability and social stigma. This chronic and debilitating condition represents one of the leading causes of preventable disability worldwide, highlighting the importance of public health measures and prompt treatment.

1.2.4. Tuberculous Lymphadenitis

Also called “scrofula,” this form of lymph node TB most commonly affects the neck region. It usually causes a gradually enlarging firm mass which, at advanced stages, eventually becomes tender and inflamed. It is characterized by a Th1 cytokine response with elevated levels of IFN-γ and TNF-α, resulting in granuloma formation [33,34]. It represents the most common form of TB outside the lungs and requires long-term antibiotic therapy.

1.2.5. Other Rare Systemic Lymphatic Conditions

Another rare lymphatic condition is called Hennekam syndrome, a genetic (autosomal recessive) disorder caused by mutations in CCBE1 and FAT4. These mutations lead to widespread lymphatic vessel malformations, which affect different parts of the body, such as the intestines, pericardium, and limbs [10,35]. Patients with this syndrome often have distinctive facial features and varying degrees of intellectual and developmental difficulties.

Yellow nail syndrome (YNS) is another uncommon disorder marked by yellow, thickened, and slow-growing nails, alongside chronic swelling (lymphedema) with frequent involvement of the respiratory system. This syndrome involves lymphatic dysfunction and sometimes low immunoglobulin levels, leading to recurrent pleural effusions and limb swelling [36]

1.3. Biomarkers in Lymphatic Disorders: Immune Signatures and Molecular Insights

The lymphatic system plays a vital role in human physiology [37]. Apart from maintaining fluid balance and absorbing dietary fats, the lymphatic system also acts as a key player in immune defense, ensuring that immune cells can circulate efficiently and respond where needed. When this system is disrupted, either due to inherited abnormalities, chronic inflammation, or malignancy, a wide variety of clinical disorders can emerge. These disorders include primary and secondary lymphedema, lymphatic malformations, lymphadenopathies, and lymphatic tumors. Therefore, there is a growing need for reliable biomarkers that can assist clinicians in better diagnosing and treating these disorders [38].

Over the past two decades, research advancements in the lymphatic system have uncovered a wide array of biomarkers. For example, immune cell patterns and cytokine profiles act as key indicators; elevated levels of IL-6, TNF-α, VEGF-C, and VEGF-D are frequently found in inflammatory or fibrotic conditions [39,40,41]. Additionally, molecular and genetic markers provide important diagnostic clues, including surface proteins like LYVE-1 [42] and D2-40 [43] or pathogenic mutations in genes such as FLT4 [44] and PIK3CA [13]. These markers have provided new insights into the epigenetics of lymphatic disorders. To better understand how these biomarkers are used in practice,

Table 1. Immune cell features, cytokine profiles, and molecular biomarkers across lymphatic disorders.

Lymphatic Disorder	Immune Cell Features	Key Cytokines	Molecular Markers	References
Primary and Secondary Lymphedema
Primary Lymphedema	CD4+ T cells (Th2 bias), M2 macrophages, impaired dendritic trafficking	TNF-α,IL-1β, IL-6, TGF-β1	VEGF-C/VEGFR-3 upregulation, TGF-b1, fibrosis markers (collagens I and III)	[7,39,40]
Milroy Disease	Abnormal lymphatic vessel development	VEGF-C, VEGFR-3	FLT4 mutation	[44]
Meige Disease	Impaired lymph drainage	VEGF-C	FOXC2 mutation	[45]
Lymphedema-Distichiasis	Distichiasis, lymphedema onset in puberty	Unknown	FOXC2	[46]
Hypoplasia of Lymphatics	Underdeveloped lymph vessels	VEGF-C	Various	[47,48]
Secondary Lymphedema	CD4+ T cells, M2 macrophages, Tregs accumulation, chronic fibrosis	IL-4, IL-6, IL-13, TGF-β, PDGF, CTGF	TGF-b, PDGF, CTGF, VEGF-C in tissue	[49,50,51]
Post-surgical Lymphedema	Local immune dysregulation	VEGF-A, TNF-α	D2-40	[43]
Post-cancer Treatment Lymphedema	T cell infiltration, fibrosis	TGF-β, IL-6	PROX1, LYVE-1	[42]
Radiation-induced Lymphedema	Chronic inflammation, macrophages	TNF-α, IL-1β	CD68	[52]
Lymphatic Filariasis	Th2 dominant, eosinophils, Tregs expansion	IL-4, IL-5, IL-10, TGF-β	CFA antigen, microfilariae, high IgE	[12,53,54]
Congenital and developmental lymphatic anomalies
Lymphatic Malformations (LMs)	Mild local inflammation, mast cells, scattered T cells (if infected)	VEGF-C, VEGF-D, IL-6, TNF-α	PIK3CA mutations, mTOR activation (pS6, pAKT)	[55,56]
Central Conduction Lymphatic Anomaly	Reduced lymphocyte recirculation, lymphocyte loss in chyle	No characteristic cytokines; possible local inflammatory mediators	Abnormal lymphatic imaging, no known genetic mutations	[57]
Generalized Lymphatic Anomaly	Macrophages and T cells around lymphatic proliferations	VEGF-C, VEGF-D, IL-6, IL-1β	PIK3CA and NRAS mutations, mTOR pathway activation, VEGFR-3, Prox1	[13,58]
Kaposiform Lymphangiomatosis	Spindle cells, mononuclear infiltration, platelet trapping	IL-6, TNF-α, elevated D-dimer related factors	NRAS mutations, VEGF, D-dimer, fibrinogen consumption	[19,20]
Intestinal Lymphangiectasia	Dilated intestinal lymphatics	Albumin loss	CD31, D2-40	[59]
Gorham–Stout Disease	Lymphatic bone invasion	VEGF-A, IL-6	RANKL	[60,61]
Cystic Hygroma	Lymph fluid-filled cysts	VEGF-C	PIK3CA	[62]
Lymphangioleiomyomatosis	-	-	VEGF-D	[63]
Lymphangiomatosis	-	-	VEGF-C/D, D2-40	[64]
Lymphadenitis and lymph node-related disorders
Lymphadenitis	Germinal center activation, granulomas (TB), T and B cell expansion	IL-1β, IL-6, IFN-g, TNF-α	PCR/culture for pathogens, high ferritin (systemic), IGRA (TB)	[65,66,67]
Castleman Disease	Plasma cell proliferation	IL-6	HHV-8 LANA	[68]
Tuberculous Lymphadenitis	Granulomas with macrophages, Langhans giant cells, strong Th1	IFN-γ, TNF-α, IL-12	Mycobacterial PCR, AFB staining, IGRA	[33]
Reactive Lymphadenopathy	Follicular hyperplasia	IL-2, IFN-γ	CD3, CD20	[69]
Autoimmune Lymphadenopathy	T/B cell hyperplasia	IL-6, TNF-α	ANA, RF	[70,71]
CVID with Lymphoid Hyperplasia	Decreased B cells	BAFF, IL-21	Low IgG	[72]
Lymphangitis	Neutrophil-dominated acute response, strong chemokine signaling	IL-1b, IL-6, TNF-α, CXCL8	Elevated CRP, procalcitonin, bacterial cultures	[73]
Neoplastic Lymphadenopathy	Clonal cell expansion	Various	BCL2, CD30	[74]
Mesenteric lymphadenitis	-	-	C-Reactive protein	[75]
Kimura Disease	-	-	Eosinophilia, serum IgE, GM-CSF, IL-4, IL-5	[76,77]
Rosai–Dorfman Disease (RDD)	-	-	Histiocytes (CD68+ve, S100+ve, CD1a-ve)	[78]
Lymphatic cancers and neoplasms
Hodgkin Lymphoma	Reed–Sternberg cells	IL-13, IL-5	CD30, CD15	[79]
Non-Hodgkin Lymphoma	B-cell or T-cell expansion	IL-6, IL-10	CD19, CD20	[80]
Lymphangiosarcoma	Endothelial malignancy	VEGF-A	CD31, ERG	[81,82]
Lymphatic metastasis	-	-	VEGF-C/VEGF-D and their receptor VEGFR-3 (FLT4)	[83,84]
Lymphoma	-	-	Pax-5, CD10, TIA-1, granzyme B, perforin, LMO2, Bcl-6, CD30, SOX-11	[85]
Rare Genetic Syndromes (with lymphatic involvement)
Turner Syndrome	Lymphedema at birth	Unknown	X chromosome abnormalities	[86]
Noonan Syndrome	Peripheral lymphedema	SHOC2 pathway	PTPN11	[87]
Yellow Nail Syndrome	Nail dystrophy, pleural effusion	-	-	[88]
Hennekam Syndrome	Possible lymphopenia, hypoalbuminemia	No clear systemic cytokines; possible local inflammatory signals	CCBE1, FAT4 mutations, Î ± 1-antitrypsin clearance	[10]

Table 1 shows lymphatic disorders by characteristic immune responses, dominant cytokine expression, and key molecular or genetic biomarkers. Conditions are grouped into categories such as primary and secondary lymphedemas, congenital and developmental lymphatic anomalies, lymphadenitis and lymph node-related disorders, lymphatic cancers and neoplasms, and genetic syndromes. Each row is supported by a peer-reviewed study, providing a reference base for both clinical and research applications. Abbreviations: AFB, acid-fast bacillus; ANA, antinuclear antibody; BAFF, B-cell activating factor; Bcl-6, B-cell lymphoma 6 protein; CD, cluster of differentiation; CFA, circulating filarial antigen; CD68, macrophage marker; CD30, TNF receptor superfamily member; CD15, granulocyte marker; CD20, B-cell marker; CD3, T-cell marker; CTGF, connective tissue growth factor; CRP, C-reactive protein; CVID, common variable immunodeficiency; CXCL8, C-X-C motif chemokine ligand 8 (also known as IL-8); D2-40, podoplanin (lymphatic endothelial marker); ERG, ETS-related gene; FLT4, Fms-like tyrosine kinase 4 (VEGFR-3); FOXC2, forkhead box protein C2; GM-CSF, granulocyte–macrophage colony-stimulating factor; HHV-8 LANA, human herpesvirus 8 latency-associated nuclear antigen; IFN-γ, interferon-gamma; IGRA, interferon-gamma release assay; IgE/IgG, immunoglobulin E/G; IL, interleukin; LMO2, LIM domain only 2; LYVE-1, lymphatic vessel endothelial hyaluronan receptor 1; mTOR, mechanistic target of rapamycin; NRAS, neuroblastoma RAS viral oncogene homolog; PDGF, platelet-derived growth factor; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; PROX1, prospero homeobox protein 1; PTPN11, protein tyrosine phosphatase non-receptor type 11; RF, rheumatoid factor; RANKL, receptor activator of nuclear factor kappa-B ligand; S100, S100 calcium-binding protein; SHOC2, soc-2 suppressor of clear homolog; SOX-11, SRY-box transcription factor 11; TIA-1, T-cell intracellular antigen 1; TGF-β, transforming growth factor beta; TNF-α, tumor necrosis factor alpha; Th1/Th2, T-helper type 1/2 cells; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.

presents an overview of the immune-related features and molecular markers found in a wide range of lymphatic disorders.

1.4. Lymphatic Remodeling in Pathological Condition

The stability of the lymphatic system is crucial for maintenance of fluid homeostasis and is dependent on proper lymph flow. Under pathological conditions of chronic inflammation and tumor metastasis, the imbalance in dynamics triggers severe outcomes. To overcome the imbalance in the fluid dynamics triggered by such pathological conditions, the lymphatic system undergoes dimensional changes (both structural and functional) to restore normal immunomodulatory functions and lymph flow. Lymphatic remodeling in common pathological conditions is summarized below.

1.4.1. Cancer-Associated Lymphatic Remodeling

The lymphatic system plays a dual role in cancer biology [89], contributing both to anti-tumor immunity and to cancer metastasis. Tumor cells secrete several growth factors or proteins which are essential for the formation of new lymphatic vessels [90]. The formation of these new lymphatic vessels provides more pathways for cancer cells to spread to other parts of the body. However, during the early stages of cancer, lymphatic vessels may carry circulating tumor antigens, which are captured and processed by dendritic cells (DCs) triggering a protective T cell response, which is then involved in the killing of tumor cells [91]. This process is important for stopping early tumor growth and is also the basis of how different immunotherapies work to prevent cancer (Figure 1). This process can be categorized into the following phases.

1.4.1.1. Lymphangiogenesis

Lymphangiogenesis is a critical pathway in cancer metastasis that favors tumor cells spreading towards the nearby lymph nodes. This process is driven by key signaling molecules, particularly vascular endothelial growth factors VEGF-C and VEGF-D, which activate receptor VEGFR-3, found mainly on lymphatic endothelial cells. Once these receptors are activated, these cells begin to grow and expand to the lymphatic network [90]. Moreover, this process also helps to create conditions favorable for tumor survival. The newly formed lymphatic vessels express immune-regulating molecules like PD-L1, which binds to PD-1 receptors present on T cells. The binding of PD-L1 to PD-1 receptors inhibits the immune response, thereby allowing the cancer cells to proliferate and expand [92].

1.4.1.2. Metastasis Through Lymphatic Vessels

In breast cancer and melanoma, the detection of lymph node metastases is associated with low survival rates and used to guide decisions on surgery and systemic therapy [93,94]. One of the key reasons cancer cells prefer lymphatic vessels is their unique structure. Lymphatic vessels have loose intercellular junctions, which further facilitate the entry of cells and fluids [95,96]. Once tumor cells enter these vessels, they can be transported to the nearby lymph nodes, which act as the first major stop in the metastatic journey. In some cases, these lymph node metastases can act as reservoirs for the cancer cells, where further distant metastases occur through the bloodstream [97]. The presence of cancer cells in lymph nodes remains an important clinical marker for staging and prognosis in many cancers, including breast carcinoma and melanoma [98].

1.4.1.3. Chemokine-Guided Tumor Migration

The dissemination of cancer cells away from the primary tumor site is a central event during metastasis process, which is regulated by various molecules, including chemokines. Chemokines are small molecules that require the immune cells to reach the inflammatory site and coordinate the immune response [99], but surprisingly many tumor cells hijack these chemokine signals and facilitate their escape and spread [95,100].

A well-studied example involves the chemokine CCL21 and its receptor CCR7, which is expressed on naïve T cells and DCs and helps them to move towards lymphoid tissues. Many tumor cells, especially in breast cancer, unusually express chemokine receptor CCR7. This helps them to relocate toward CCL21 production sites such as lymphatic vessels and lymph nodes. [100,101]. Another chemokine CXCL12/SDF-1 (stromal cell-derived factor 1) and its receptor CXCL12 also play a prominent role in tumor dissemination. Moreover, a previous study has shown that neutralization of CXCL12-CXCR4 axis interaction significantly compromised the tumor cells’ dissemination [102]. In addition, the transient expression of CXCR4 level increased in the presence of hypoxia and VEGFs in the tumor microenvironment [103]. From a clinical perspective, small molecular inhibitor of CXCR4 such as plerixafor or BKT140 have been shown to facilitate the transfer of hematopoietic stem cells into the bloodstream during autologous transplant in multiple myeloma [104].

1.4.1.4. Lymphatics in Anti-Tumor Immunity

Although the lymphatic system is highlighted for its role in cancer metastasis, it also plays a crucial role in anti-tumor immunity by serving as a pathway for immune cell trafficking and antigen presentation. This process begins with immature DCs, which capture and process soluble tumor antigens within the tumor microenvironment. Once loaded with these antigens, DCs undergo maturation and migrate to tumor-draining lymph nodes, where they present these tumor antigens on major histocompatibility complex (MHC) molecules to naïve T cells and initiate a protective T-cell response [105]. For effective T-cell activation, the interaction between MHC and T-cell receptors (TCRs) is not enough as it requires a series of co-stimulatory signals, including CD80/86 molecules, on DCs engaging with CD28 receptors on T cells [106]. These adaptive immune signals result in the infiltration of activated CD8⁺ cytotoxic T (Tc) cells into the tumor, where they directly kill tumor cells.

The role of lymphatics in cancer therapy has significantly improved over the last decade. For instance, cancer vaccines depend on healthy lymphatic vessels to carry antigens to nearby lymph nodes, where they can trigger strong T cell responses [107]. In a similar manner, treatments like immune checkpoint inhibitors such as anti-PD-1 or anti-CTLA-4 antibodies also rely on a well-functioning connection between the lymphatic system and the immune system to help activate and spread tumor-targeting T cells.

1.4.1.5. Immunosuppression Within Tumor-Draining Lymph Nodes

Tumor-draining lymph nodes (TDLNs) are essential in helping the immune system to detect and respond against cancer. In many cancers, TDLNs become altered in ways that help the tumor grow and spread by promoting an immunosuppressive environment [108,109]. This change begins when the tumor cells send signals to the lymph node, where the structure and function of the lymph node are reshaped. These signals recruit and upregulate the differentiation of Tregs, regulatory B cells (Bregs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs), all of which work to weaken the immune response [110,111]. Moreover, DCs inside the TDLN become more tolerogenic, expressing low levels of co-stimulatory molecules CD80 and CD86 [112]. Cytokines such as TGF-β, IL-10, and IDO (indoleamine 2,3-dioxygenase) further suppress this, creating an environment that shuts down normal immune functions [113,114]. Even when tumor-specific T cells are present, they often become exhausted. These exhausted T cells express several immune checkpoint markers, including PD-1, TIM-3, and LAG-3, which reduce their proliferation and cytokine production, impairing tumor cell killing [115,116].

1.5. Clinical and Translational Perspectives

The dual role of the lymphatic system in promoting both anti-tumor immunity and inducing metastasis has clinical implications for cancer immunotherapy. Immune checkpoint blockade therapy such as anti-PD-1 and anti-CTLA-4 mainly rely on the proper activation and migration of T cells. Since lymphatic vessels and TDLNs are central components to this immune activation process, their structural and functional integrity are essential for treatment success [117]. Recent studies showed that preserving TDLNs during surgery may improve responses to immunotherapy. These nodes house reservoirs of stem-like or pre-exhausted T cells, which can expand and migrate to tumor sites during checkpoint blockade therapy [118]. The removal of these lymph nodes prematurely, before or during immunotherapy, may reduce the pool of responsive T cells and compromise treatment outcomes. Moreover, targeting tumor-induced lymphangiogenesis by blocking VEGF-C/VEGFR-3 not only inhibits metastatic spread, but also reverses immunosuppressive remodeling of the lymphatic environment [90,98]. Through better understanding how the lymphatic system works in cancer, we can explore new ways to improve immunotherapy, make more informed choices during surgery and develop combined treatments that boost the body’s immune response while limiting metastasis.

In addition to structural and cellular remodeling of the lymphatic system, a broad range of molecular and genetic biomarkers orchestrate immune activation and tumor-associated immune evasion. These biomarkers include inflammatory cytokines, chemokines, immune checkpoints, lymphangiogenic growth factors, and key oncogenic signaling regulators that collectively shape the tumor-draining lymphatic microenvironment.

Table 2. Biomarkers in anti-tumor immunity and cancer immune evasion.

Category	Biomarker	Functional Role in Tumor Immunity/Evasion	Major Pathways/Mechanisms	References
Inflammatory Cytokines	IL-6	Promotes inflammation, tumor growth, and immune suppression	JAK/STAT3, NF-κB	[119,120]
	TNF-α	Triggers inflammation: chronic levels support invasion	NF-κB, MAPK	[119,120]
	TGF-β	Induces Tregs and suppresses effector T cells	SMAD, PI3K/AKT	[120,121]
	IL-10	Inhibits antigen presentation and T-cell activity	STAT3, SOCS3	[122,123]
Pro-Immunogenic Cytokine	IFN-γ	Activates cytotoxic T cells, upregulates MHC, induces PD-L1	JAK/STAT1, IRF1	[119,124]
	IL-2	Stimulates proliferation and activation of cytotoxic T cells and NK cells	JAK/STAT5, PI3K/AKT	[120]
	IL-12	Promotes Th1 differentiation, IFN-γ production, and cytotoxic immune response	STAT4, NF-κB	[119,120]
	IL-15	Enhances NK cell and CD8+ T-cell survival and memory formation	JAK1/3–STAT5, PI3K/AKT	[125]
	IL-18	Synergizes with IL-12 to boost IFN-γ release and antitumor activity	MyD88, NF-κB, MAPK	[126]
Chemokine Axes	CCL21/CCR7	Guides immune cells; hijacked by tumors for LN migration	Chemokine, MAPK	[119]
	CXCL12/CXCR4	Mediates tumor homing and lymphatic metastasis	PI3K/AKT, ERK	[120]
Immune Checkpoints	PD-1	Suppresses T-cell activation upon PD-L1 binding	PD-1/PD-L1 axis	[127,128]
	PD-L1	Inhibits cytotoxic T cells; induced by hypoxia and IFN-γ	JAK/STAT, HIF-1α	[127,128]
	CTLA-4	Blocks T-cell priming and proliferation	CD80/86–CTLA-4	[120,129]
	LAG-3	Restrains T-cell proliferation under chronic antigen load	LAG-3/MHC-II	[120,130,131]
	TIM-3	Promotes T-cell exhaustion and tolerance	TIM-3/Gal-9	[120,132]
Lymphangiogenic/Angiogenic Factors	VEGF-C	Induces lymphangiogenesis and metastasis	VEGF-C/VEGFR-3	[133]
	VEGF-D	Works with VEGF-C to remodel lymphatics	VEGFR-3	[133]
	VEGF-A	Stimulates angiogenesis and vascular permeability	VEGF-A/VEGFR-2	[134]
Lymphatic Markers	LYVE-1	Marker of active lymphatics; linked to metastasis	Lymphatic endothelial	[119]
	D2-40 (Podoplanin)	Marker of lymphatic invasion; enhances motility	Rho-GTPase, PI3K	[135]
Genetic/Oncogenic Pathways	FLT4 (VEGFR-3)	Mediates VEGF-C/D signaling; promotes metastasis	PI3K/AKT, MAPK	[136]
	PIK3CA	Activates PI3K pathway; drives lymphatic proliferation	PI3K/AKT/mTOR	[120]
	PTEN	Tumor suppressor; loss enhances immune evasion	PI3K/AKT	[120]
	HIF-1α	Hypoxia-induced PD-L1 and VEGF expression	HIF-1α/VEGF	[127,128]
	mTOR	Integrates growth and immune signals	PI3K/AKT/mTOR	[120]

Table 2 Summary of key biomarker molecules involved in anti-tumor immunity and cancer cell evasion of immune surveillance. The table lists representative molecular and genetic markers, including cytokines, chemokines, immune-checkpoint molecules, lymphangiogenic factors, and signaling genes that contribute to the regulation of immune activation and tumor immune escape. Abbreviations: AKT, protein kinase B; CCR7, C-C chemokine receptor 7; CXCR4, C-X-C chemokine receptor 4; DC, dendritic cell; EMT, epithelial–mesenchymal transition; HIF-1α, hypoxia-inducible factor 1α; IFN-γ, interferon-γ; IL, interleukin; JAK, Janus kinase; LAG-3, lymphocyte activation gene-3; LYVE-1, lymphatic vessel endothelial hyaluronan receptor 1; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor kappa B; PD-1/PD-L1, programmed death receptor 1/ligand 1; PI3K, phosphatidylinositol-3-kinase; PTEN, phosphatase and tensin homolog; SMAD, mothers against decapentaplegic homolog; STAT, signal transducer and activator of transcription; TAM, tumor-associated macrophage; TGF-β, transforming growth factor β; TIM-3, T-cell immunoglobulin and mucin-domain containing-3; TNF-α, tumor necrosis factor α; Treg, regulatory T cell; VEGF, vascular endothelial growth factor; VEGFR-3 (FLT4), vascular endothelial growth factor receptor-3.

summarizes the key biomarkers and their pathways associated with anti-tumor immunity and immune escape.

1.5.1. Lymphedema Remodeling

Lymphedema also described above, is just not an obstruction of proper lymph flow or drainage characterized by fluid accumulation but also fat deposition in the tissues. Disruption in fluid transport is triggered by hypertrophic (increase in size) fat lobules, hindering lymphatic capillaries and further leading to fat accumulation in tissues. Also observed in obesity, the remodeling of adipose tissue such as hypertrophy and hyperplasia (increase in size) in lymphedema induces metabolic stress and increased pro-inflammatory polarization through cytokines and chemokines like IL-6, TNF-$\alpha$, MCP-1, and IL-8 [137,138,139,140]. The dilation of lymphatic vessels and fibrosis in the subcutaneous tissue and lymphatic vessel wall have also been observed in lymphedema. The accumulation of excess collagen impairs vessel contraction, resulting in the hardening of tissues and disruptions in lymph flow [138].

1.5.2. Autoimmune-Associated Remodeling

Rheumatoid Arthritis (RA) is often linked with lymphangiogenesis and increased vessel contraction to remove inflammatory cells and cellular debris at the inflamed site. Hypertrophy of draining lymph nodes is also observed in response to the inflamed environment. Similar to lymphedema, increased collagen deposition and vesicular dilation is linked to systemic sclerosis. Lymphangiogenesis is also observed in some systemic lupus erythematosus (SLE) conditions [141].

1.6. Therapeutic Targets: Targeting Lymphatic Remodeling

Lymphatic remodeling during pathological conditions is of great interest for therapeutic targets in recent times. Identifying key molecular targets driving lymphangionegensis (VEGF-C/D), lymphatics development (PROX1), vessel dilation and contraction (nitric oxide signaling and cytokines such as IL-6, TNF-α, are being utilized as therapeutic interventions in lymphedema and cancer [142]. Studies also suggest that tumor cell-derived extracellular vesicles contribute to metastatic lymphatic remodeling and can be a therapeutic target [143].

1.7. Tertiary Lymphoid Structures

In addition to secondary lymphoid organs, the lymphatic system contributes to the formation of specialized immune niches called tertiary lymphoid structures (TLSs). TLSs are ectopic lymphoid structures in non-lymphoid tissues and are formed spontaneously in response and proximity to persistent inflammation or injury (autoimmunity, cancers, transplant rejection, gliomas, etc.) unlike lymphoid organs, which begin to develop early in the embryonic life of the fetus. TLSs are often referred as iBALT (inducible bronchus-associated lymphoid tissue) in the lung and are induced by air pollutants like ozone, asbestos fiber exposure, autoimmune conditions, and allergies [144,145,146]. The number, shape, and cellular composition of TLSs vary with the different physiological conditions and locations [147]. Because of their proximity to an inflamed environment, TLSs serve as a local reservoir that is important for adaptive immunity to maintain tissue homeostasis. Although the regulation of immune cell recruitment is largely unknown, as TLSs continue to mature in an inflamed environment, the cellular phenotype also adapts accordingly.

1.7.1. Cellular Composition of TLS

Generally, mature TLSs consists of a central zone of B cells surrounded by T cells in the periphery and high endothelial venules (HEVs) [148]. The peripheral T cell zone is dominated by T cell subsets of cytotoxic (Tc), helper T cells (Th), and follicular T cells (Tfh) [149]. Depending on the nature of their environment, antigen presenting cells (APCs) like macrophages, neutrophils, and DCs can also be found in these structures. In lung cancer, the TLS is heterogenous, ranging from disorganized cellular aggregates to well-organized structures containing follicles dominated by T cells, mature DCs, and follicular B cells. Studies also suggest that the TLS functioning as an antitumor immunity consists of DCs, B cells, Tfh cells, chemokines, and lymphoid tissue-inducer (LTI) cells [149,150,151]. The presence of LTI cells promotes the production of signaling cytokines, adhesion molecules, and integrins to attract adaptive cells in the TLS area [152].

1.7.2. Regulatory Mechanisms Related to Immunology

The development of the TLS starts with lymphocyte aggregation. This step in TLS formation does not include germinal centers and a separate zone for B and T cells. As the TLS gradually matures, T and B cells start to form well-organized areas with expanded follicular DCs in the germinal center [153]. TLSs are complicated aggregates of immune cells formed by a complex network of functionally redundant cytokines, which are heterogenous in different pathological conditions. Cytokines of the interleukin family, such as IL-4, IL-6, IL-7, IL-33 etc., chemokines such as CXCL12, CXCL13, CCL21, etc., interferons such as IFN-$\gamma$, Type 1 IFN, etc., TNFs, and TGF-$\beta$ promote the formation and functioning of the TLS [153,154,155,156]. Interleukins primarily function through modulating the proliferation and differentiation of B, Tfh, and Th17 cells. Chemokines recruit B, T, and DCs, promoting the formation of B and T cell zones whereas interferons facilitate HEV formation, Tfh differentiation, and B-cell activating factor (BAFF) expression for B cell development and function. HEVs maintain immune surveillance and trafficking through promoting the migration of immune cells to TLSs through the bloodstream. TNFs activate fibroblasts and the expression of chemokines and adhesion molecules. Taken together, these interrelated mechanisms promote TLSs enriched with activated immune cells and signaling molecules as a form of protective immunity in pathological disease conditions.

1.7.3. Metabolic Reprogramming in TLSs

Since TLSs are formed in response to a maladaptive environment, the metabolism of immune cells also differs between the cells in the lymphoid tissues and the TLS. The pathological environment demands the metabolic reprogramming of immune cells so that the TLS can work efficiently and adapt to the inflamed environment. For instance, activated T cells induce rapid aerobic glycolysis for differentiation and cytokine production [157]. In contrast, T cells within the TLS may undergo either reduced or altered glycolysis in response to the pathological microenvironment. This shift can suppress the activation of IgG-producing B cells and memory B cells [158]. Apart from glycolysis, activated T and B cells can also engage glutaminolysis, oxidative phosphorylation, and fatty acid oxidation pathways [151] for energy generation, proliferation, expansion, and differentiation. On the other hand, T and B cells might experience reprogramming of these pathways for energy generation to further facilitate anti-tumor immunity. In the tumor environment, tumor cells outcompete the normal metabolism of immune cells for nutrients essential for tumor growth. Metabolites like lactic acid are known to be generated in the tumor environment, which promote acidosis [159], suppressing immune cell function. This further might promote immunosuppressive function by enhanced Tregs differentiation. Metabolic reprogramming and lymphatic remodeling can also exhaust T cell function by promoting tissue hypoxia [159,160]. Hypoxia is generally promoted by tumor cells rapidly proliferating and outcompeting the normal immune cell metabolism.

1.7.4. TLS, Its Association with Diseases, and Therapeutic Advances

TLSs can have both advantages and disadvantages based on the pathological conditions. Studies suggest that in diseases like cancer, the presence of TLSs is often linked with better prognosis [153,161]. In contrary, TLS is linked with poor prognosis in autoimmune diseases [162]. Tumor environment promoting the metabolic reprogramming of immune cells can trigger an environment which can be inflammatory. The metabolic pathways driving an inflammatory phenotype triggering chronic outcomes have been extensively studied over the last decade as therapeutic targets [151]. To enhance the efficacy of antitumor therapy, several strategies to induce TLSs, such as vaccines, immune checkpoint blockades (ICB), antiangiogenesis, and CAR-T cell therapy, are being studied. Several studies highlight that TLS induction increases the immunotherapy response in cancer patients after ICB therapy [148,153,158,163]. Another strategy of activating effector Tc cells, depleting Tregs, is shown to promote T cell trafficking and HEVs’ formation [148,164,165].

2. Techniques and Advances in Prognosis and Therapy

The advances in molecular imaging, lymphatic surgery, and strategies for lymph node-targeted nano-delivery have significantly helped in assessment, intervention, and understanding therapeutic approaches associated with lymphatic disorders. Preventing the progression of lymphedema and enhancing patient outcomes requires an early and precise diagnosis. This can be made possible through the combined skills of nuclear medicine and radiology, followed by possible surgical handling. Together with conventional drug administration, the scientific community is now more focused on targeted drug delivery using nanoparticles to increase the efficacy and safety of vaccines and immunotherapies.

2.1. Advances in Molecular Imaging

The imaging technique has evolved substantially since the beginning of the modern era of lymphatic imaging in 1952, from pedal lymphography by Kinmonth [166] to the currently prevalent modern cross-sectional imaging using ultrasonography (US) and computed tomography (CT).

2.1.1. Lymphoscintigraphy (LS)

The most conventional technique, X-ray lymphography, was modified by Sherman and Ter-Pogossian into lymphoscintigraphy [167], which remained a gold standard nuclear medicine modality for decades in confirming the diagnosis of lymphedema. As part of the procedure, a tracer-labeled large molecule is injected into the tissues, and the lymphatic transport function can be characterized by imaging the signal that is released using a gamma camera. This technique lacks standardization [168] and has poor spatial and temporal resolution. However, for the diagnosis of lymphedema, it has high reported sensitivity (96%) and specificity (100%) [169]. The lack of anatomic information in this technique has been enhanced through combination with single-photon emission computerized tomography/computed tomography (SPECT/CT) [170], which helps in precise, pre-operative SLN mapping to assist surgical planning in multiple types of cancer [171,172,173].

2.1.2. Fluorescence Imaging

Fluorescence microlymphography uses FITC-dextran, which is injected intradermally, and the tracer is visualized using a microscope [174]. This technique was rarely adopted by lymphologists as FITC-dextran did not receive official approval for human use. Near-infrared (NIR) lymphography is a comparatively new technique which uses clinically approved indocyanine green (ICG) for intradermal injections near the area of interest and images these using a coupled-charged detector (CCD) camera [175]. Compared to lymphoscintigraphy, it promised improved spatial and temporal resolution. It added a precise quantitative analysis of collecting vessel contractility [176,177], and was thus very useful in assessing lymphatic flow in the transplants [178,179]. This technique helped in identifying lymphatic channels before LVA surgery [180], and mapping lymphatic vessels in cancer patients [181]. Their poor stability, self-quenching nature, and iodine content restricts the applicability of these tracers [182].

2.1.3. Ultrasonography (US)

Ultrasonography excludes the venous components of lymphedema and is helpful in determining suitable conditions for lympho-venous anastomoses (LVA). More in-depth examinations can be performed using high-frequency (HFUS) and ultra-high-frequency (UHFUS) probes [183,184]. The most recent sonographic tool, contrast-enhanced ultrasonography (CEUS), can detect sentinel lymph nodes and lymphatic vessels [185,186]. In many cases CEUS, HFUS, and UFHUS are improved alternatives to indocyanine green (ICG) lymphography and effective tools for planning LVA procedures, revealing potential venules, the best optimal regions, and lymphatic vessels [187,188].

2.1.4. Magnetic Resonance Lymphography (MRL)

MRL is a highly sensitive technique in confirming the diagnosis of lymphedema and adipose hypertrophy. Without radiation exposure, it shows the location of lymphatics and the magnitude of dermal backflows, and can be used to detect occult metastases and venous blockage in 3D imaging [189,190]. Non-contrast MRL emphasizes signals from accumulated fluid and suppresses signals from solid tissues. It uses heavily T2-weighted sequences, which help in the effective visualization of central lymphatics [191]. The contrast-enhanced MRL adds the interstitial injection of a gadolinium (Gd)-based tracer over non-contrast MRL, and the enrichment signal is sensed on T1-weighted images. The blood vessels and lymphatic vessels absorb the Gd-based MR contrast agent, making it difficult to differentiate between these vascular systems. However, the anatomical differences results in the delayed uptake in lymphatics compared to blood vasculature, which can be visualized using a series of dynamic images [192]. Despite these benefits, the costs, extensive exposure time, absence of lymphatic specific tracers, and risk of contrast allergies discourage its utility.

2.1.5. Computed Tomography

CT plays a supportive role in lymphedema diagnostics. It is useful in locating perforated vessels for lymphatic tissue transfer [193]. It can assess the excess amount of fibrous tissue and detect the severity of edema but cannot distinguish between lymphedema and edema [190].

2.1.6. Photoacoustic Imaging (PAI)

PAI is a promising imaging technique which combines optical absorption and ultrasound detection. It uses short pulse, non-ionizing lights, which are absorbed by the tissue to generate heat and thermal expansion to create ultrasound waves [194]. Even in the areas of dermal backflow where the NIRF camera only detects splash or diffuse ICG deposition, PAI enables the precise visualization of lymphatic vessels without any ionizing radiation exposure [195]. PAI is very scalable and has a very high resolution. The photoacoustic microscopy has low depth penetration with high resolution, while photoacoustic tomography has a larger depth penetration but a lower resolution [196,197,198]. The use of Evans blue dye has significantly expanded the potential and application of PAI [199]. Scientists have evaluated the potential of several dyes, such as Evans blue and ICG, and found it comparable to NIR imaging [200,201].

2.1.7. Optical Coherence Tomography (OCT)

OCT provides label-free 3D reconstruction of optical scattering within tissues. Using this technique, it has been demonstrated that lymphatic tubes are visible due to the negative contrast of the surrounding tissue, which is highly scattered. These lymphatic tubes may be further distinguished from blood vessels as there are fewer intraluminal cells [202,203,204]. Using a microscope-assisted OCT system, mice treated with sutures to stimulate inflammatory lymphangiogenesis could be used to render lymphatic vessels within their corneas [205]. Another group used this technique to visualize conjunctival lymphatic vessels in porcine eyes ex vivo [206]. Further studies will evaluate the potential of OCT in imaging of ocular lymphatic vessels.

2.2. Nanobiotechnology in Molecular Imaging

Nanoparticle (NP)-based contrast agents tagged with fluorescent dyes or radioactive isotope augment the visibility of cancer cells in imaging techniques such as PET, MRI, and CT scans [207,208]. This development is helpful in the early detection of metastasis to improve treatment outcomes. The biocompatibility and excellent photothermal property of gold NPs (AuNPs) make them an ideal candidate [209]. Semiconductor-based NPs like quantum dots (QDs) provide high fluorescence and stability and allow for long-term imaging [210]. To increase targeted accuracy, NPs can be attached with ligands or antibodies that attach to certain tumor markers [211,212]. The development of nanobiotechnology tools in molecular imaging has been reviewed by Wu et al. [213].

2.3. Advances in Lymph Node-Targeted Nano-Delivery

Tumor cells metastasize TDLNs and release substances such lactic acid and extracellular vesicles (EVs), resulting in decreased T-cell activity and the development of Tregs [97,214,215]. Remodeling the immunosuppressive microenvironment of TDLNs is necessary to reinstate the T cell activity and augment immunotherapy efficacy. Targeted delivery using nanoparticles (NPs) could be a promising solution to address these challenges.

Nanoparticles offer the efficient and precise targeted delivery of therapeutic agents to LNs [216,217]. These technologies have excellent biocompatibility, with easy surface modification and high tissue permeability [218]. Several factors determine the recognition and interaction of NPs with the cell surface and endocytosis pathway when administered in vivo. These include the route of delivery and physical properties like particle shape, size, surface charge, hydrophilicity, and bioactivity [219,220].

2.3.1. Route of Administration

Other than oral administration, the lymphatic targeting of NPs includes intranodal, interstitial, or intravenous injections. Intranodal injection has been the most effective approach in eliciting robust immune response, but technical challenges and the complexity of the administration process limit its applicability [221,222]. Interstitial injections are the most common clinical practice. The intravenously administered NPs are absorbed by the mononuclear phagocyte system and accumulate in the kidneys or liver before moving into lymphatic arteries and interstitial tissues [223]. Although NPs can be modified to increase circulation time, ineffective LN targeting still limits systemic delivery.

2.3.2. Physical Properties

The study by Vania Manolova et al. determined that NPs (20–200 nm) could directly internalize the LNs parenchyma through the subcapsular sinus (SCS) and accumulate in B-cell areas within 2 h when injected into the footpads of mice [224]. This approach was further enhanced with the use of biodegradable and programmable surface linkers with NPs, resulting in quick release upon LN targeting and allowing for their interaction with more lymphocyte subtypes [225].

Positively charged NPs are more easily ingested by APCs but suffer from higher cytotoxicity and reduced target efficiency [226]. The neutral and negatively charged NPs have shown improved LN accumulation [227,228]. Charge-reversal materials could potentially be an effective way to get around the drawbacks of surface charge and improve cellular absorption [229,230]. Hydrophobic NPs are more easily absorbed by APCs; hydrophilic NPs are efficiently transported across water channels [231,232]. The hydrophilic molecule polyethylene glycol (PEG) revolutionized NPs delivery, with improved stability [233,234]. Further, De Koker et al. developed PEG-modified PMA NPs (PEG-PMA) with enhanced LNs accumulation in comparison to non-PEGylated PMA NPs [235]. Different immune cells respond differently to mechanical changes and altering the deformability of NPs can drastically influence the efficiency of their cellular absorption and the mechanism of endocytosis [236]. Several molecules, like mesenchymal stem cell membrane-encapsulated silica NPs (MCSNs) [237], deformable albumin-stabilized emulsions [238], and NPs (Lipo-NPs) with adjustable elasticity [239], have been developed to enhance cellular uptake and interaction between NPs and the immune system.

Ligands allow NPs to either “hitchhike” on migratory APCs for transfer into LNs or activate resident APCs. The development of glucosylated nanovaccines for neo-antigen delivery by Liu et al. significantly improved antigen delivery [240]. APC-mediated LN targeting has been enhanced by targeting CLEC9A [241], DEC-205 [242], and mannose receptors [243]. NPs have been conjugated with antibodies like CD11b [244] and CD11c [245] to facilitate their uptake and transfer to lymph nodes. The accumulation of NPs in LNs is also influenced by SCS macrophages, the first line of defense forming a barrier against LNs. Zhang et al. demonstrated that the transport and accumulation of ovalbumin-conjugated spherical gold nanoparticles (OVA-AuNPs) in LNs follicles were markedly improved by the preemptive reduction in SCS macrophages in mice following the intradermal injection of clodronate liposomes [246].

Biofilm-encapsulated nanoparticles provide outstanding biocompatibility, an extended circulation period, and functional integration through cell membrane proteins [247]. The membrane of mature DCs [248], macrophages [249], and tumor cells [250] have shown promising results. Another biomimetic technique in NPs is the use of EVs, which are nanoscale lipid bilayer vesicles released by cells [251,252,253]. Physical modification, the ligand-based approach, biofilm-coated NPs, and EVs-based strategies have demonstrated significant potential in improving immune activation, LN accumulation, and targeted delivery. In conclusion, the rational design of NPs, guided by the physiological structure of LNs to effectively navigate or bypass barriers and achieve precise drug accumulation, holds great potential for further improving the efficacy of tumor therapy.

2.4. Current Advances in AI-Assisted Diagnosis

Artificial Intelligence (AI) has become an integral part of imaging analysis in health care services. The patient care system is now utilizing the domains of AI, like machine learning (ML), robotics, fussy model (FM), and natural language processing (NLP) [254]. ML arises at the intersection of statistics by learning relationships from data using efficient computing algorithms [255]. Supervised ML can be used to automate ECG and X-ray image to reach a diagnosis. The use of robotics in surgery is growing with the integration of robotic staging and robotic-assisted surgery in standard practice [256,257]. NLP is helpful in developing chatbots that communicate with patients to provide health care services.

Vicentini et al. presented the first model, which employed an FM to objectively classify the risk and severity of lymphedema [258]. The model quantitatively evaluates the lymphedema stage, taking class overlap into account using a set of “IF-THEN” fuzzy rules. This model admits varying degrees of severity for each input and allows for overlap within classes, resulting in both qualitative and quantitative outcomes. Later, three ML algorithms were tested by Moreira et al. to preclinically detect lymphedema in breast cancer survivors, but the best performance ended up showing 19% inaccuracy [259]. Recent studies have demonstrated promising results in predicting lymph node metastasis in rectal cancer. The MRI-based AI diagnosis performed on par with radiologist diagnoses, while CT-based AI performed better [260,261,262]. The lymph node metastases diagnostic model (LNMDM) developed by Wu et al. effectively detected tumor metastasis, particularly micro-metastasis [263]. AI models based on radiomics and DL have the potential to predict lymph node metastasis more accurately. However, the clinical implementation of these models requires more deep learning studies and multicenter tests due to heterogeneity in radiomics studies [264,265,266,267]. Besides imaging data, blood test and therapy data have also been effectively utilized to develop ML models for the early detection of lymphedema, with more reliable outcomes compared to the patient-self-reporting data system [268].

Robotic-assisted surgery is well known in gynecologic oncology and provides a safe and efficient method for endometrial and cervical cancer staging and treatment [257]. Retrospective research that compared the results of open surgery and robotic-assisted hysterectomy for cervical cancer reported a significant decrease in the incidence of postoperative lower-limb lymphedema [256]. A similar comparison for endometrial cancer supports a decrease in the incidence of postoperative lymphedema [269]. Moreover, there is still a lack of solid evidence from randomized clinical trials. Two recent cases from Cleveland clinic reported a first-of-its-kind robotic-assisted microsurgery. One of them is the first robot-assisted super microsurgical lymphatic venous bypass for breast cancer-related lymphedema of the arm and the other is the world’s first robotically assisted lymphatic venous bypass to treat lymphedema in breast [270].

Challenges and Future Perspectives

Molecular imaging faces several challenges in the accurate detection of lymph node metastasis. The higher cost restricts its wider accessibility and affordability. Sensitivity and specificity are still the fundamental concern [271]. The utilization of new biomarkers and tracers specific to cancer tissues can enhance the sensitivity of detection. Micro-metastasis, an important event that warrants early detection, often remains ignored in conventional imaging techniques, and thus innovative advances are desirable for effective detection [272,273]. Proper differentiation between metastatic and reactive lymph nodes is essential for accurate staging and treatment planning. The integration of molecular imaging with other modalities necessitates the use of AI and machine learning, involving sophisticated data analysis techniques.

The use of NPs and nanoconjugates demonstrate significant promise in the identification and management of lymph node metastases; however, this has several drawbacks. The physical and biological barriers, as well as the complex microenvironment, remain the major challenge in the efficient delivery of NPs to the lymph nodes. Lymph node targeting could be maximized with careful engineering of NPs’ shape, size, and surface characteristics. The development of a universal NP or nanoconjugate is a challenge due to variability in the location, size, and level of lymph node metastasis [274]. A thorough assessment of the biodistribution, pharmacokinetics, and safety profile of NPs and nanoconjugates is crucial before clinical translation. Further research is required for the development of safer and effective NP-based strategies.

There are significant challenges associated with the prediction of malignant lymph nodes with currently available machine learning tools. The variations in imaging protocol, imaging parameters, type of scanner used, image format, and different scanning techniques throughout institutions create discrepancies in image quality and dampen the model’s ability to efficiently analyze new data sets. Consistent AI performance across various clinical set-ups requires standardization of preanalytical data elements for biospecimens [275]. Swarm learning is one decentralized learning approach being investigated in cancer histology to address issues related to data privacy and facilitate a broader and more cooperative utilization of AI models across institutions [276]. The performance of AI models depends on training data, and thus the models can learn and perpetuate biases available in the data, resulting in performance differences between various demographic groups [277,278]. A fair and equitable outcome must be ensured to rule out these demographic biases in computational pathology.

Many of the deep learning models operate as “black boxes”, and it might be challenging to understand the reasoning behind their predictions. Research into explainable AI (XAI) is being intensively explored to improve the interpretability and transparency of these models to help doctors better understand the AI-driven judgements. There are ethical issues pertaining to diagnostic errors and the responsibilities assigned in the course of the development and application of AI in pathology. These challenges must be addressed by negotiations with intricate regulatory frameworks like FDA approval (US) and IVDR (Europe). Institutions like the Ecosystem for Pathology Diagnostic with AI Assistance (EMPAIA) are working to develop guidelines to help resolve these issues [279,280].

3. Conclusions

The lymphatic system plays a central role in fluid balance, nutrient absorption, and immune defense. Dysfunction of this system leads to lymphatic disorders, which can be congenital or acquired. Lymphatic remodeling in pathological conditions is essential to restore the proper function of the lymph flow. Moreover, cancer-associated lymphatic remodeling plays a dual role. It can spread the tumor cells through lymphatic vessels by a process called lymphangiogenesis. Furthermore, chemokines and their receptors play a potential role during the metastasis process. In addition, the generation of tolerogenic DCs and TAMs, and the secretion of immunosuppressive molecules such as IL-10, TGFβ, and IDO, also promote tumor growth. In contrast, the lymphatic system plays a crucial role in anti-tumor immunity by involving APCs such as DCs and Tc cells. This review highlighted a comprehensive overview of the lymphatic system, including their disorders, with remodeling during pathological conditions being more focused on cancer and ectopic TLSs. The last section of this review includes therapeutic advances in imaging, nanotechnology, and nano-delivery with the integration of artificial intelligence, together with challenges and future perspectives.