Interrogating the roles of lymph node metastasis in systemic immune surveillance

Abstract

Lymph nodes (LNs) are principal orchestrators of the adaptive immune response, yet in the context of malignancy, they are typically the first sites of metastasis. When tumors spread to LNs, they alter the immune repertoire, ultimately reconditioning it in a manner that suppresses anti-tumor immunity and promotes further metastatic dissemination. Conversely, activation of anti-tumor immunity within LNs is essential for immunotherapy, suggesting clinical approaches to radiotherapy in LNs and lymphadenectomy may need to be reconsidered in the context of immune checkpoint blockade (ICB). Herein, we discuss our understanding of the immune remodeling that coincides with LN metastasis as well as recent clinical studies exploring neoadjuvant immunotherapy and the roles of LNs in treatment of solid organ malignancies.

Introduction

Metastatic spread to distant organs leads to the majority of cancer-related deaths [1], yet the mechanisms promoting dissemination and colonization remain unclear. Prior to metastasis, genomic alterations, inherited or acquired through environmental insults, underpin malignant transformation. The mutated proteins, when expressed, can serve as neoepitopes, potentiating recognition by the adaptive immune system. Thus, while the acquisition of mutations is necessary for neoplasia and the formation of tumors, they render the tumors vulnerable to clearance by the immune system. To overcome this vulnerability, tumors acquire additional traits that endow them with the capacity to locally suppress immune responses or to evade them entirely [2]. Nonetheless, while disseminating throughout the body in the absence of such local microenvironmental suppressive features, individual tumor cells are exposed to the full breadth of the immune response, rendering the process of metastasis an inefficient process. Thus, the anti-tumor adaptive immune response represents a particularly potent barrier to the process of metastasis, perhaps more so than it does to growth of the primary tumor.

Previous preclinical investigations employing mouse models of metastasis have greatly advanced our understanding of the mechanisms underlying distant metastatic spread, but the majority of these discoveries have been conducted in immune compromised murine models [3]. Nonetheless, the advent of the immunotherapy era in cancer therapy has made it imperative for future evaluations of metastatic models to be conducted in immune competent hosts.

Lymph node involvement has traditionally been a harbinger of distant metastatic disease and has been incorporated in staging, prognostication, and treatment decision making in clinical oncology [4]. Key to orchestrating a robust adaptive immune response, the lymph node is a critical secondary lymphoid organ that serves as an education hub where naïve lymphocytes encounter antigen presenting cells (APCs), follicular dendritic cells, and lymphoid cells, and subsequently learn to recognize new antigens, transition to effector cells, and eventually develop immunologic memory [5]. Lymph nodes are highly compartmentalized and organized structures which serve as barriers against systemic dissemination of disease. The context in which antigen is presented within lymph nodes is a key determinant of whether that antigen will be recognized by the immune system as pathogenic or benign. Pathogens, such as Mycobacterium tuberculosis and Yersinia pestis, have been shown to coopt mechanisms of tolerance to evade immunity [6, 7]. Similarly, tumors may also utilize this existing architecture to actively hinder an effective anti-tumor immune response thereby enabling further metastatic dissemination.

A range of mouse and human studies have implicated roles for tumor conditioning of immune responses in lymph nodes prior to their colonization of those nodes. These processes include contributions from non-immune stroma such as lymphatic endothelial cells and various fibroblasts and have been reviewed in detail elsewhere [8–13]. We focus this review on changes in the immune compartment of the tumor-draining lymph node following metastatic colonization and the subsequent effects on the systemic immune response.

Models of lymph node metastasis

Until recently, there existed two predominant models that describe the routes by which cancer spreads throughout the host. The Halstedian model describes serial metastatic progression wherein lymph nodes represent an intermediate metastatic site and serve as a staging ground for the subsequent seeding of distant metastases [14, 15]. Indeed, studies in mice have demonstrated that lymph node tumors can seed distant sites [15, 16]. Conversely, genomic reconstruction of metastatic phylogenies from genome sequencing or sequencing of hypermutable regions gave rise to an alternate model known as the Systemic Model. This model proposed that the majority of distant metastases are clonally distinct from lymph node metastases, although typically harboring the same driver mutations, suggesting parallel spread from a heterogenous primary tumor [17]. Thus, the systemic model suggests that lymph node colonization plays no functional role in tumor progression. Recently, we developed a murine model of lymph node metastasis, which led to the generation of a new conceptual framework for metastasis known as Metastatic Tolerance (Fig. 1). We discovered that by colonizing lymph nodes, tumors induce immune tolerance through the induction of antigen-specific regulatory T cells (Tregs). This tolerance subsequently becomes systemic, rendering distant sites amenable to metastatic colonization [18]. Thus, distant metastases do not necessarily need to be derived from lymph node metastases, but colonization of lymph nodes is a critical step in metastatic progression due to its importance in the generation of immunological tolerance.

Fig. 1.

Immune interactions during LN metastasis. Primary tumors colonize LNs, wherein they condition both the innate and adaptive arms of the immune response to promote tumor progression. In particular the induction of tumor-specific regulatory T cells (Tregs) supports metastasis to distant tissues

Effects of lymph node metastasis on the myeloid compartment within the lymph node

Under normal physiological conditions, the myeloid compartment consists of less than 5% of total cells within the lymph node, comprising a combination of dendritic cells (DCs), macrophages, and neutrophils. Using our murine model of LN metastatic potential of melanoma, we previously interrogated alterations within these populations in response to LN metastasis [18]. DCs play essential roles in the education of T cells, orchestrating the balance between their activation and suppression. Within tumor-involved lymph nodes, overall DC numbers were unaltered by the presence of LN metastases, but the DC subpopulation composition shifted towards an increase of resident MHCII^intCD11c^hi dendritic cells (rDCs) in comparison to MHCII^hiCD11c^int migratory DCs (mDCs) and a reduction in cross-presenting Xcr1⁺ classical dendritic cells (cDC1). Notably, there were increased numbers of CD11b⁺ DCs, particularly CD11b⁺CD8a⁻ cells, which predominantly encompass cDC2s. These CD11b⁺SIRPα⁺ cDC2s express much higher levels of co-inhibitory molecule Programmed Death-Ligand 1 (PD-L1) both in mice bearing only primary tumors and those with LN metastases suggesting that their relative increase in frequency may reflect a shifting balance towards immunosuppression [19]. Slight changes in dendritic cell repertoire from tumor draining lymph nodes can significantly impact immune responses. For example, a prior study suggested that a small population of indoleamine 2,3-dioxygenase 1 (IDO1)-expressing plasmacytoid DCs from the tumor draining lymph node was capable of inducing Treg generation, T cell anergy, and decreased T cell responses to tumor antigens [20]. Additionally, dendritic cells have been reported to exhibit dysfunctional or decreased antigen presentation in patients with tumors [21, 22]. Other studies have suggested that Tregs within tumor draining lymph nodes (tdLNs) inhibit the generation of anti-tumor immunity by cDC2s migrating from primary tumor to the LNs [23]. Further studies into which cell type is the major contributor towards generation of this immunosuppressive phenotype are needed.

Macrophages within the lymph node compartment are divided into subcapsular sinus macrophages (F4/80⁻CD169⁺), medullary sinus macrophages (F4/80⁺CD169⁺), and medullary cord macrophages (F4/80⁺CD169⁻) [24]. Subcapsular sinus macrophages are typically the first immune cells encountered by foreign pathogens, antigens, and tumors after they enter the lymph node via lymphatics. The density of CD169⁺ macrophages in tumor draining lymph nodes has been positively associated with favorable prognosis in locally advanced colorectal and breast cancers [25, 26]. In our study, within tumor involved lymph nodes, there were increased total cell numbers and frequencies of neutrophils and macrophages. Notably, macrophages within the tumor-involved lymph nodes had significantly higher levels of PD-L1 compared to those in LNs of mice bearing only primary tumors or in healthy nodes, suggesting a transition towards a suppressive phenotype. Gene expression of neutrophils revealed signatures consistent with immature and immunosuppressive phenotypes. In a study of human LNs involved with gastric adenocarcinoma, there was an increase in neutrophils bearing a tumor-associated markers (CD54^low) [27].

Effects of lymph node metastasis on the lymphoid compartment within the lymph node

The lymphoid compartment is the predominant immune population within lymph nodes, comprising approximately 95% of immune cells. LNs are the site of B and T cell education and maturation. Studies in patients with a variety of solid organ malignancies have revealed associations between LN metastasis and increases in CD4⁺CD25⁺ regulatory T cells (Tregs) [28–31]. Similarly, in our murine melanoma LN metastasis model, in comparison to primary tumor, we have demonstrated that there is a decrease in T:B cell ratio and an overall increase in FoxP3⁺CD25⁺CD4⁺ Tregs in mice bearing LN metastatic tumors. To determine whether these Tregs were tumor antigen specific, they were isolated from the LNs of mice bearing ovalbumin-expressing tumors. MHCII-OVA tetramer staining revealed that mice bearing the LN metastatic tumors had a higher fraction of Tregs that recognize the tumors. Furthermore, the Tregs from mice bearing LN metastases promoted higher levels of lung metastasis when adoptively transferred to mice challenged with tail vein injections of tumor compared with Tregs from mice with no LN involvement. Using mice bearing transgenic TCRs that did not recognize the tumors, we also found that antigen specificity of the Tregs TCR was required for the enhanced promotion of distant metastasis in the context of LN involvement.

In addition to alterations in the Treg compartment, LN metastasis influences several other lymphocyte subsets. There were significantly increased frequencies of CD44^hiCD62L⁻ memory and effector CD8⁺ cells and increased PD-1 expression on CD8⁺ T cells within tumor involved lymph nodes, potentially indicative of exhaustion phenotypes. Although NK cells have been reported to increase in number within lymph nodes during viral and bacterial infections, their role in lymph node tumor metastases is incompletely understood. While loss or expression or alterations in NKG2D ligands may confer tumor escape from NK cells [32], our mouse model revealed that upregulation of MHC-I is an early and essential adaptation for some tumors to metastasize to LNs. This upregulation was conserved in patients and additional mouse models of pancreatic adenocarcinoma, head and neck cancer, and melanoma [18]. Cytotoxic NK cells have been shown to accumulate in tumor involved lymph nodes from melanoma patients [33], indicating that overcoming this first line of defense against metastases is a critical step for metastatic progression of tumors. Thus, the findings from our studies and others suggest that once this barrier is overcome, tumors can exploit the immune priming environment of the lymph node to reprogram the antigen-specific immune response to support tumor progression.

Clinical implications

It has long been appreciated that patients lacking LN involvement exhibit superior prognoses compared with those bearing LN metastases. Consequently, treatment of locally advanced solid malignancies for curative intent typically involved removal of the primary tumor and regional lymph nodes in combination with chemotherapy, radiation, or both. The benefit of surgical lymphadenectomy has been controversial due to risks of increased morbidity from postoperative adverse side effects such as chronic lymphedema as well as conflicting data regarding whether extensive locoregional LN removal improves overall survival in comparison to adjuvant chemotherapy, radiation, or both [34–37]. In the context of immunotherapy, efficacy is now attributed to activation of systemic immune responses within LNs rather than reinvigoration of tumor-localized immune cells, calling into question whether lymphadenectomy may inhibit the generation of anti-tumor immunity [38–43], though this effect may be ameliorated with a neoadjuvant approach.

Several clinical trials in solid tumors demonstrate improved pathologic complete response or progression free survival with a neoadjuvant approach, defined typically as a combination chemotherapy and immunotherapy, prior to surgery, for locally advanced resectable tumors. Neoadjuvant immune checkpoint blockade with dostarlimab, a PD-1 inhibitor, in mismatch repair deficient (dMMR) rectal cancer recently demonstrated complete response in all 12 patients thereby bypassing the need for surgery or definitive radiation, although long term survival is still being evaluated [44]. These neoadjuvant approaches are now, or soon to be, FDA approved as standard of care, Table 1. The rationale for a neoadjuvant or perioperative approach are (1) retention of tumor-involved LNs, which are critical in educating antigen-specific anti-tumor immune responses required to eradicate occult metastatic disease, (2) the presence of an intact primary tumor providing a rich source of tumor antigens [40], and (3) preservation of an immune repertoire that has not been depleted by the effects of chemotherapies or radiation [45]. In perioperative treatment, immunotherapy is typically administered both before surgery, to prime the immune system, and after surgery, as the majority of randomized clinical trials are not designed with arms comparing neoadjuvant (prior to surgery) or perioperative immunotherapy in comparison to adjuvant (after surgery) treatment. The requisite duration of adjuvant immunotherapy remains unclear as most checkpoint trials are not designed to evaluate cessation of treatment. Therefore, there may be opportunities for new immunotherapy non-inferiority trials to shorten adjuvant immunotherapy durations. A recent phase 2 clinical trial in resectable Stage 3B to 4C melanoma demonstrated significant event free survival at 14.7 months for the neoadjuvant-adjuvant pembrolizumab arm 72% [95% CI 64 to 80] compared to 49% [95% CI 41 to 59] in the adjuvant pembrolizumab alone [46].

Table 1.

Select clinical trials of neoadjuvant immunotherapy for resectable disease

Tumor type (citation)	Stage	Number of patients (randomization)	Arms	Findings
Triple negative breast carcinoma [61]	2, 3	602 (2:1)	4 cycles of pembrolizumab vs. placebo q3w + carboplatin and paclitaxel → 4 cycles of pembrolizumab vs. placebo → ddACT → surgery → pembrolizumab or placebo up to 9 cycles	64.8% immunotherapy [95% CI, 59.9 to 69.5] vs. 51.2% placebo [95% CI, 44.1 to 58.3] pCR
dMMR gastric adenocarcinoma [62]	2,3	32	Single arm neoadjuvant ipilimumab q6w × 2 + nivolumab q3w	58.6% had pCR, 90% CI, 41.8 to 74.1
dMMR rectal adenocarcinoma [44]	2, 3	12	Single arm dostarlimab q3w for 6 months	100% complete response
Melanoma [46]	3B to 4C	313 (1:1)	Neoadjuvant (3 doses) → surgery → adjuvant (15 doses) pembrolizumab q3w vs. surgery → adjuvant alone (up to 18 doses)	72% [95% CI, 64,80] vs. 49% [95% CI, 41,59] EFS at 14.7 months
Non-small cell Lung Adenocarcinoma [63]	2, 3A, 3B	397 (1:1)	Pembrolizumab vs. placebo q3w + cisplatin based chemotherapy followed by surgery followed by adjuvant pembrolizumab vs. placebo q3 weeks up to 13 cycles	62.4% immunotherapy vs. 40.6% placebo EFS at 24 months HR 0.58 [95% CI 0.46 to 0.72]; 18% vs 4% pCR
Non- small cell lung adenocarcinoma [64]	3	86 (2:1)	Nivolumab + platinum based chemotherapy → surgery → adjuvant nivolumab if R0 vs. chemotherapy alone → surgery	37% neoadjuvant-adjuvant vs. 7% adjuvant pCR (RR = 5.34; [95% CI, 1.34 to 21.23]
Cutaneous squamous cell carcinoma [65]	2,3,4	79	Single arm cemiplimab q3w for 4 cycles → surgery	51% pCR [95% CI, 39 to 62]
Glioblastoma [66]	Recurrent resectable	35 (1:1)	Neoadjuvant pembrolizumab 2 weeks prior to surgical resection followed by adjuvant pembrolizumab vs. surgery followed by adjuvant pembrolizumab	417 days neoadjuvant-adjuvant vs. 228 days adjuvant alone median OS

pCR pathologic complete response, dMMR deficient mismatch repair, EFS event free survival, CI confidence interval, HR hazard ratio, OS overall survival, RR relative risk, q3w every 3 weeks, q6w every 6 weeks

Given the importance of LNs in generating anti-tumor immunity and the benefits of neoadjuvant ICB, the extent of LN dissection beyond sentinel LN biopsy, for diagnostic purposes, remains an active area of debate warranting additional trials. Preclinical studies employing pharmacologic inhibition of T cell egress or lymphadenectomy of tumor draining LNs have demonstrated a decrease in checkpoint inhibitor response and CD8+ tumor infiltration [40, 42, 43]. Consideration of which LNs should be retained may be specific to tumor types, molecular signatures, or both. Notably, there may be a threshold of metastatic burden in which the tumor within the LN serves as a potent reservoir of metastatic cells with limited immune priming capability, and removal after perioperative immunotherapy would be advised. Additionally, there is evidence that expansion of B cells in tertiary lymphoid structures is seen in patients with melanomas [47], renal cell carcinomas [48], and dMMR rectal adenocarcinomas [44] that respond to immune checkpoint blockade and the role of LN metastatic tolerance in relation to these processes remains to be elucidated. Notably, the liver is an immune privileged site, and there is increasing evidence that patients with liver metastases are less responsive to immune checkpoint blockade in non-small cell lung adenocarcinoma [49], melanoma, and other cancers [50, 51]. Many patients with liver metastases also exhibit LN involvement, however, and determining the interplay between these two tolerance mechanisms warrants further investigation. Even prior to LN colonization, conditioning of the immune response by tumor-draining lymphatics likely plays an important role in immune suppression [52, 53]. In light of the importance of LNs in facilitating clinical responses to immunotherapy, advancement in drug delivery systems specifically targeting lymph nodes to assist immune priming or reverse tolerance may provide benefit in halting further dissemination of metastatic disease [54, 55].

While our understanding of the mechanisms by which LN metastases alter immune responses remains in its infancy, insights might be gleaned from studying advanced stage lymphomas, where the malignant lymphocytic cells by nature home to LNs. In classic Hodgkin’s lymphoma, high PD-L1 expression is seen in both the tumor and lymph node [56]. Our data in solid tumors showed discordance between a higher PD-L1 expression in LN metastases compared to a lower expression seen in primary and distant metastases in both murine and human samples, which may explain why clinical responses can occur in patients receiving immune checkpoint therapy targeting the PD-L1/PD1 axis in the absence of PD-L1 surface expression from the biopsied primary tumor specimen [57–60], Fig. 2. Notably, in clinical decision making, the tissue analyzed for PD-L1 expression by immunohistochemistry could be derived from the primary tumor, lymph node metastases, or distant organ metastases, and these different tissues within a single patient can have significant variations in PD-L1 expression. Whether events leading to increased PD-L1 expression are a consequence of tumor drainage [40] without tumor cell contact in the lymph node is yet to be determined. Thus, while both the tumor-intrinsic and immune mechanisms by which LN metastases promote further progression and immune suppression warrant further investigation, it is clear that LNs play essential roles both in facilitating metastatic progression and mounting robust anti-tumor immunity during treatment with immunotherapy. We believe that future clinical approaches to immunotherapy, radiotherapy, and lymphadenectomy would be aided by careful consideration of their implications within the conceptual framework of LNs as orchestrators of immune responses to tumors.

Fig. 2.

Hypothetical explanations for discordance between PD-L1 immunohistochemical analysis of biopsies and anti-PD-1/PD-L1 treatment response

Data availability

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.