Lymphatic system regulation of anti-cancer immunity and metastasis.

Cancer dissemination to lymph nodes (LN) is associated with a worse prognosis, increased incidence of distant metastases and reduced response to therapy. The LN microenvironment puts selective pressure on cancer cells, creating cells that can survive in LN as well as providing survival advantages for distant metastatic spread. Additionally, the presence of cancer cells leads to an immunosuppressive LN microenvironment, favoring the evasion of anti-cancer immune surveillance. However, recent studies have also characterized previously unrecognized roles for tumor-draining lymph nodes (TDLNs) in cancer immunotherapy response, including acting as a reservoir for pre-exhausted CD8+ T cells and stem-like CD8+ T cells. In this review, we will discuss the spread of cancer cells through the lymphatic system, the roles of TDLNs in metastasis and anti-cancer immune responses, and the therapeutic opportunities and challenges in targeting LN metastasis.

The transcription regulator ID3 maintains tumor-specific memory CD8+ T cells in draining lymph nodes during tumorigenesis.

During tumorigenesis, the recently identified tumor-specific memory T cells in draining lymph nodes (TdLN-TTSM cells) play a pivotal role in tumor repression that gives rise to progenitor exhausted T (TPEX) cells and further replenishes tumor-specific CD8+ T cells residing in the tumor microenvironment (TME). However, how TTSM cells are maintained in TdLN is largely unknown. Here, we show that the transcription regulator ID3 (inhibitor of DNA binding 3) is highly expressed by TTSM cells compared with other CD8+ T cell subsets. The deficiency of ID3 significantly interrupts the maintenance of TTSM and TPEX cells, resulting in decreased tumor-infiltrating CD8+ T cells and impaired tumor control. Consistent with this, overexpression of ID3 in CD8+ T cells increases the TTSM cell population and enhances the anti-tumor immune response.

Microbiota-induced plastic T cells enhance immune control of antigen-sharing tumors.

Therapies that harness the immune system to target and eliminate tumor cells have revolutionized cancer care. Immune checkpoint blockade (ICB), which boosts the anti-tumor immune response by inhibiting negative regulators of T cell activation1-3, is remarkably successful in a subset of cancer patients, yet a significant proportion do not respond to treatment, emphasizing the need to understand factors influencing the therapeutic efficacy of ICB4-9. The gut microbiota, consisting of trillions of microorganisms residing in the gastrointestinal tract, has emerged as a critical determinant of immune function and response to cancer immunotherapy, with multiple studies demonstrating association of microbiota composition with clinical response10-16. However, a mechanistic understanding of how gut commensal bacteria influence the efficacy of ICB remains elusive. Here we utilized a gut commensal microorganism, segmented filamentous bacteria (SFB), which induces an antigen-specific Th17 cell effector program17, to investigate how colonization with it affects the efficacy of ICB in restraining distal growth of tumors sharing antigen with SFB. We find that anti-PD-1 treatment effectively inhibits the growth of implanted SFB antigen-expressing melanoma only if mice are colonized with SFB. Through T cell receptor clonal lineage tracing, fate mapping, and peptide-MHC tetramer staining, we identify tumor-associated SFB-specific Th1-like cells derived from the homeostatic Th17 cells induced by SFB colonization in the small intestine lamina propria. These gut-educated ex-Th17 cells produce high levels of the pro-inflammatory cytokines IFN-γ and TNF-α, and promote expansion and effector functions of CD8+ tumor-infiltrating cytotoxic lymphocytes, thereby controlling tumor growth. A better understanding of how distinct intestinal commensal microbes can promote T cell plasticity-dependent responses against antigen-sharing tumors may allow for the design of novel cancer immunotherapeutic strategies.

Preclinical study and phase II trial of adapting low-dose radiotherapy to immunotherapy in small cell lung cancer.

BACKGROUND: Immune checkpoint inhibitors (ICIs) provide modest but unsatisfactory benefits for extensive-stage small cell lung cancer (ES-SCLC). Developing strategies for treating ES-SCLC is critical. METHODS: We preliminarily explored the outcomes of salvage low-dose radiotherapy (LDRT) plus ICI on refractory SCLC patients. Next, we evaluated the combinational efficacy in murine SCLC. The tumor immune microenvironment (TIME) was analyzed for mechanistic study. Subsequently, we conducted a multicenter, prospective phase II trial that administered concurrent thoracic LDRT plus chemoimmunotherapy to treatment-naive ES-SCLC patients (MATCH trial, NCT04622228). The primary endpoint was confirmed objective response rate (ORR), and the key secondary endpoints included progression-free survival (PFS) and safety. FINDINGS: Fifteen refractory SCLC patients treated with LDRT plus ICI were retrospectively reviewed. The ORR was 73.3% (95% confidence interval [CI], 44.9-92.2). We identified a specific dose of LDRT (15 Gy/5 fractions) that exhibited growth retardation and improved survival in murine SCLC when combined with ICIs. This combination recruited a special T cell population, TCF1+ PD-1+ CD8+ stem-like T cells, from tumor-draining lymph nodes into the TIME. The MATCH trial showed a confirmed ORR of 87.5% (95% CI, 75.9-94.8). The median PFS was 6.9 months (95% CI, 5.4-9.3). CONCLUSIONS: These findings verified that LDRT plus chemoimmunotherapy was safe, feasible, and effective for ES-SCLC, warranting further investigation. FUNDING: This research was funded by West China Hospital (no. ZYJC21003), the National Natural Science Foundation of China (no. 82073336), and the MATCH trial was fully funded by Roche (China) Holding Ltd. (RCHL) and Shanghai Roche Pharmaceuticals Ltd. (SRPL).

Intra-tumoral administration of CHST15 siRNA remodels tumor microenvironment and augments tumor-infiltrating T cells in pancreatic cancer.

The dense stroma is one cause of poor efficacy of T cell-mediated immunotherapy in pancreatic ductal adenocarcinoma (PDAC). Carbohydrate sulfotransferase 15 (CHST15) is a proteoglycan-synthetic enzyme responsible for remodeling tumor stroma. Intra-tumoral injection of CHST15 small interfering RNA (siRNA) has been shown to increase the tumor-infiltrating T cells (TILs) in patients with unresectable PDAC. However, the mechanism underlying the enhanced accumulation of TILs is not fully explored. Here, we demonstrate that intra-tumoral injection of CHST15 siRNA locally and remotely diminishes myeloid-derived suppressor cells (MDSCs) and enhances TILs in mice. CHST15 was expressed by tumor cells and MDSCs in both tumor and tumor-draining lymph nodes (TDLNs), and CHST15 siRNA repressed stromal density, neutrophil extracellular traps, and Ly6C/G+ MDSCs in vivo. Remarkably, tumor growth inhibition was only observed in the immunocompetent KPC model, which is associated with enhanced TILs. In vitro, CHST15 siRNA significantly downregulated the levels of CHST15 and indoleamine 2,3-dioxygenase mRNA in CD33+ MDSCs derived from human peripheral blood mononuclear cells. These results suggest a dual role for intra-tumorally injected CHST15 siRNA on modulating the tumor immune microenvironment for T cell entry and remotely diminishing CHST15+ MDSCs, decreasing T cell suppression and expanding T cells in the TDLN, ultimately leading to an enhanced accumulation of TILs.

Enhanced oral versus flank lymph node T cell response parallels anti-PD1 efficacy in head and neck cancer.

OBJECTIVES: Understanding head and neck tissue specific immune responses is important for elucidating immunotherapy resistance mechanisms to head and neck squamous cell carcinoma (HNSCC). In this study, we aimed to investigate HNSCC-specific immune response differences between oral and subcutaneous flank tumor transplantation in preclinical models. MATERIALS AND METHODS: The MOC1 syngeneic mouse oral carcinoma cell line or versions expressing either the H2Kb-restricted SIINFEKL peptide from ovalbumin (MOC1OVA) or ZsGreen (MOC1ZsGreen) were inoculated into mouse oral mucosa (buccal space) or subcutaneous flank and compared for immune cell kinetics in tumors and tumor-draining lymph nodes (TDLNs) and for anti-PD1 response. RESULTS: Compared to subcutaneous flank tumors, orthotopic oral MOC1OVA induced a higher number of OVA-specific T cells, PD1 + or CD69 + activated OVA-specific T cells in both primary tumors and TDLNs. Tumors were also larger in the flank site and CD8 depletion eliminated the difference in tumor weight between the two sites. Oral versus flank SIINFEKL peptide vaccination showed enhanced TDLN lymphocyte response in the former site. Notably, cDC1 from oral TDLN showed enhanced antigen uptake and co-stimulatory marker expression, resulting in elicitation of an increased antigen specific T cell response and increased activated T cells. Parental MOC1 in the oral site showed increased endogenous antigen-reactive T cells in TDLNs and anti-PD1 blockade rejected oral MOC1 tumors but not subcutaneous flank MOC1. CONCLUSION: Collectively, we find distinct immune responses between orthotopic oral and heterotopic subcutaneous models, including priming by cDC1 in TDLN, revealing important implications for head and neck cancer preclinical studies.

CD4+ T cell activation distinguishes response to anti-PD-L1+anti-CTLA4 therapy from anti-PD-L1 monotherapy.

Cancer patients often receive a combination of antibodies targeting programmed death-ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen-4 (CTLA4). We conducted a window-of-opportunity study in head and neck squamous cell carcinoma (HNSCC) to examine the contribution of anti-CTLA4 to anti-PD-L1 therapy. Single-cell profiling of on- versus pre-treatment biopsies identified T cell expansion as an early response marker. In tumors, anti-PD-L1 triggered the expansion of mostly CD8+ T cells, whereas combination therapy expanded both CD4+ and CD8+ T cells. Such CD4+ T cells exhibited an activated T helper 1 (Th1) phenotype. CD4+ and CD8+ T cells co-localized with and were surrounded by dendritic cells expressing T cell homing factors or antibody-producing plasma cells. T cell receptor tracing suggests that anti-CTLA4, but not anti-PD-L1, triggers the trafficking of CD4+ naive/central-memory T cells from tumor-draining lymph nodes (tdLNs), via blood, to the tumor wherein T cells acquire a Th1 phenotype. Thus, CD4+ T cell activation and recruitment from tdLNs are hallmarks of early response to anti-PD-L1 plus anti-CTLA4 in HNSCC.

Radiation and anti-PD-L1 synergize by stimulating a stem-like T cell population in the tumor-draining lymph node.

Radiotherapy (RT) and anti-PD-L1 synergize to enhance local and distant (abscopal) tumor control. However, clinical results in humans have been variable. With the goal of improving clinical outcomes, we investigated the underlying synergistic mechanism focusing on a CD8+ PD-1+ Tcf-1+ stem-like T cell subset in the tumor-draining lymph node (TdLN). Using murine melanoma models, we found that RT + anti-PD-L1 induces a novel differentiation program in the TdLN stem-like population which leads to their expansion and differentiation into effector cells within the tumor. Our data indicate that optimal synergy between RT + anti-PD-L1 is dependent on the TdLN stem-like T cell population as either blockade of TdLN egress or specific stem-like T cell depletion reduced tumor control. Together, these data demonstrate a multistep stimulation of stem-like T cells following combination therapy which is initiated in the TdLN and completed in the tumor.

Immune changes in hilar tumor draining lymph nodes following node sparing neoadjuvant chemoradiotherapy of localized cN0 non-small cell lung cancer.

Background: While much progress has been accomplished in the understanding of radiation-induced immune effects in tumors, little is known regarding the mechanisms involved at the tumor draining lymph node (TDLN) level. The objective of this retrospective study was to assess the immune and biological changes arising in non-involved TDLNs upon node sparing concurrent chemoradiotherapy (CRT) of non-small cell lung cancer (NSCLC) tumors. Methods: Patients with proven localized (cN0M0) NSCLC, treated by radical surgery plus lymph node dissection with (CRT+) or without (CRT-) neoadjuvant chemoradiotherapy, whereby radiotherapy was targeted on the primary tumor with no significant incidental irradiation of the non-involved TDLN station (stations XI), were identified. Bulk RNA sequencing of TDLNs was performed and data were analyzed based on differential gene expression (DGE) and gene sets enrichment. Results: Sixteen patients were included and 25 TDLNs were analyzed: 6 patients in the CRT+ group (12 samples) and 10 patients in the CRT- group (13 samples). Overall, 1001 genes were differentially expressed between the two groups (CRT+ and CRT-). Analysis with g-profiler revealed that gene sets associated with antitumor immune response, inflammatory response, hypoxia, angiogenesis, epithelial mesenchymal transition and extra-cellular matrix remodeling were enriched in the CRT+ group, whereas only gene sets associated with B cells and B-cell receptor signaling were enriched in the CRT- group. Unsupervised dimensionality reduction identified two clusters of TDLNs from CRT+ patients, of which one cluster (cluster 1) exhibited higher expression of pathways identified as enriched in the overall CRT+ group in comparison to the CRT- group. In CRT+ cluster 1, 3 out of 3 patients had pathological complete response (pCR) or major pathological response (MPR) to neoadjuvant CRT, whereas only 1 out of 3 patients in the other CRT+ cluster (cluster 2) experienced MPR and none exhibited pCR. Conclusion: Neoadjuvant node sparing concurrent CRT of NSCLC patients is associated with distinct microenvironment and immunological patterns in non-involved TDLNs as compared to non-involved TDLNs from patients with non-irradiated tumors. Our data are in line with studies showing superiority of lymph node sparing irradiation of the primary tumor in the induction of antitumor immunity.

The prognostic value of the histological shape of tumor negative sentinel nodes in breast cancer.

Introduction: Sentinel lymph node (SLN) metastasis is an important predictor of prognosis in breast cancer (BC) patients, guiding treatment decisions. However, patients with the same BC subtype and tumor negative SLN (SLNneg) can have different survival outcomes. We hypothesized that the host anti-tumor immune reaction in SLNneg is important and results in morphometrically measurable changes in SLN size or shape which are related to patient prognosis. Methods: Surface area, circumference, long axis and short axis were histologically measured in 694 SLNneg from 356 cases of invasive BC and 67 ductal carcinoma in situ cases. The area occupied by fat was categorized as less or more than 50%. The long to short axis (L/S) ratio was calculated. The relationship between SLNneg morphometries and clinicopathological variables like tumor-infiltrating lymphocytes (TILs) within the primary tumor, as well as prognosis at 10 years follow up were analyzed. Results: The mean SLNneg surface area was 78.7mm2, circumference 40.3mm, long axis 13.1mm, short axis 8.2mm and L/S ratio 1.7. Larger surface area, long axis and short axis, including age >55 years were associated with higher body mass index (BMI) and SLN fat over 50% (p<0.003). In invasive BC, a high SLNneg L/S ratio (≥1.9) was related to poorer disease-free (HR=1.805, 95%CI 1.182-2.755, p=0.006) and overall (HR=2.389, 95%CI 1.481-3.851, p<0.001) survival. A low SLNneg L/S ratio (<1.9) was associated with high TILs in the primary BC (≥10%) (p=0.005). However a high TIL count was not of prognostic relevance. Conclusions: This is the first study to suggest that morphometric characteristics of axillary SLNneg, like L/S ratio, could be used to predict prognosis in patients with SLNneg invasive BC of all subtypes. The association between low L/S ratio and high TILs suggest that SLN shape is related to immunological functioning of the SLN and could be used in addition to TIL evaluation. Regarding the dubious role of TILs in hormone receptor positive breast cancer, SLNneg morphometry to gain information about host immune status could especially be of benefit in this subtype. Further studies are warranted to better understand the underlying biological mechanisms.

Immunomodulatory properties of the lymphatic endothelium in the tumor microenvironment.

The tumor microenvironment (TME) is an intricate complex and dynamic structure composed of various cell types, including tumor, stromal and immune cells. Within this complex network, lymphatic endothelial cells (LECs) play a crucial role in regulating immune responses and influencing tumor progression and metastatic dissemination to lymph node and distant organs. Interestingly, LECs possess unique immunomodulatory properties that can either promote or inhibit anti-tumor immune responses. In fact, tumor-associated lymphangiogenesis can facilitate tumor cell dissemination and metastasis supporting immunoevasion, but also, different molecular mechanisms involved in LEC-mediated anti-tumor immunity have been already described. In this context, the crosstalk between cancer cells, LECs and immune cells and how this communication can shape the immune landscape in the TME is gaining increased interest in recent years. In this review, we present a comprehensive and updated report about the immunomodulatory properties of the lymphatic endothelium within the TME, with special focus on primary tumors and tumor-draining lymph nodes. Furthermore, we outline emerging research investigating the potential therapeutic strategies targeting the lymphatic endothelium to enhance anti-tumor immune responses. Understanding the intricate mechanisms involved in LEC-mediated immune modulation in the TME opens up new possibilities for the development of innovative approaches to fight cancer.

[Impact of immunotherapy on the therapeutic strategy for the management of stage I non-small cell lung cancer: The radiation oncologist's point of view]. / Influence de l'immunothérapie sur le choix du traitement des cancers bronchiques non à petites cellules de stade I : point de vue de l'oncologue radiothérapeute.

Surgery is the standard treatment for operable patients with stage I non-small cell lung cancer (NSCLC) (T1-T2aN0M0). Stereotactic body radiotherapy (SBRT) is the treatment of choice for non-operable patients, and its positioning for operable patients remains to be clarified. The pattern of recurrence after management of stage I NSCLC is dominated by the risk of distant recurrence, this constituting the rationale for the adjunction of systemic treatment, and especially check point inhibitor (CPI), in combination with surgery or SBRT for patients with high risk features. While the benefit of postoperative CPI on the micro-metastatic disease is logically considered within the framework of a simply additive effect of both therapeutic modalities, it is reasonable to consider a synergistic effect of both CPI and SBRT. Given the role of tumor draining nodes in the development of an anti-tumor immune response, a "tumor-draining node sparing" strategy enabled by SBRT could therefore be of major interest in combination with CPI. Pending confirmation of the role of CPI in combination with RTS for the management of stage I NSCLC, we thus discuss in this review the theoretical advantages that this therapeutic strategy could have compared to a surgical strategy.

Tumor immunogenicity dictates reliance on TCF1 in CD8+ T cells for response to immunotherapy.

Stem-like CD8+ T cells are regulated by T cell factor 1 (TCF1) and are considered requisite for immune checkpoint blockade (ICB) response. However, recent findings indicate that reliance on TCF1+CD8+ T cells for ICB efficacy may differ across tumor contexts. We find that TCF1 is essential for optimal priming of tumor antigen-specific CD8+ T cells and ICB response in poorly immunogenic tumors that accumulate TOX+ dysfunctional T cells, but is dispensable for T cell priming and therapy response in highly immunogenic tumors that efficiently expand transitory effectors. Importantly, improving T cell priming by vaccination or by enhancing antigen presentation on tumors rescues the defective responses of TCF1-deficient CD8+ T cells upon ICB in poorly immunogenic tumors. Our study highlights TCF1's role during the early stages of anti-tumor CD8+ T cell responses with important implications for guiding optimal therapeutic interventions in cancers with low TCF1+CD8+ T cells and low-neo-antigen expression.

A Novel Method Using Fine Needle Aspiration from Tumor-Draining Lymph Nodes Could Enable the Discovery of New Prognostic Markers in Patients with Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell cancer (cSCC) is the second most common form of skin cancer, characterized by abnormal, accelerated growth of squamous cells. When caught early, most cSCCs are curable. About 5 percent of the cSCC cases have advanced to such an extent, generally metastatic, that they are far more dangerous, with very poor prognosis and challenging to treat. All efforts to find biomarkers, in blood or in the tumor itself, for early identification of patients with a risk for metastasis have so far failed. The present study describes a novel method that enables the identification of lymphocyte markers in tumor-draining lymph nodes. Six patients with advanced cSCC were analyzed using a combination of a sentinel lymph node biopsy (SLNB) protocol, fine needle aspiration (FNA), and flow cytometry. Immunological results from the sentinel nodes were combined with corresponding data from peripheral blood and unfixed tumor tissues. The result demonstrates a striking difference between the subsets of T-cells from the three compartments. Our interpretation of this first pilot study is that the ability to follow specific immunological markers on lymphocytes in tumor-draining lymph nodes will enable the identification of novel prognostic biomarkers not detectable in material from blood and tumor tissues.

Combination therapy with hydrogen peroxide and irradiation promotes an abscopal effect in mouse models.

Hydrogen peroxide (H2 O2 ) induces oxidative stress and cytotoxicity, and can be used for treating cancers in combination with radiotherapy. A product comprising H2 O2 and sodium hyaluronate has been developed as a radiosensitizer. However, the effects of H2 O2 on antitumor immunity remain unclear. To investigate the effects of H2 O2 , especially the abscopal effect when combined with radiotherapy (RT), we implanted murine tumor cells simultaneously in two locations in mouse models: the hind limb and back. H2 O2 mixed with sodium hyaluronate was injected intratumorally, followed by irradiation only at the hind limb lesion. No treatment was administered to the back lesion. The H2 O2 /RT combination significantly reduced tumor growth at the noninjected/nonirradiated site in the back lesion, whereas H2 O2 or RT individually did not reduce tumor growth. Flow cytometric analyses of the tumor-draining lymph nodes in the injected/irradiated areas showed that the number of dendritic cells increased significantly with maturation in the H2 O2 /RT combination group. In addition, analyses of tumor-infiltrating lymphocytes showed that the number of CD8+ (cluster of differentiation 8) T cells and the frequency of IFN-γ+ (interferon gamma) CD8+ T cells were higher in the noninjected/nonirradiated tumors in the H2 O2 /RT group compared to those in the other groups. PD-1 (programmed death receptor 1) blockade further increased the antitumor effect against noninjected/nonirradiated tumors in the H2 O2 /RT group. Intratumoral injection of H2 O2 combined with RT therefore induces an abscopal effect by activating antitumor immunity, which can be further enhanced by PD-1 blockade. These findings promote the development of H2 O2 /RT therapy combined with cancer immunotherapies, even for advanced cancers.

Silencing of tumoral carbohydrate sulfotransferase 15 reactivates lymph node pancreatic cancer T cells in mice.

Limited intratumoral T-cell infiltration in pancreatic ductal adenocarcinoma (PDAC) is an obstacle to immunotherapy, yet the efficient approach to enhance tumor-infiltrating T cells is not fully established. Here, we show that tumor-specific knockdown of carbohydrate sulfotransferase 15 (CHST15), a tumor stromal proteoglycan-synthetic enzyme, suppresses tumor growth in a T-cell-dependent manner in a murine model of PDAC. Silencing of tumoral CHST15 unexpectedly expanded CD4+ and CD8+ T cells in tumor draining LN (TDLN), leading to accelerated accumulation of EdU+ proliferating CD4+ and CD8+ T cells and granzyme B+ CD8+ T cells in the tumor. RNA expression analysis indicated that tumoral CHST15 knockdown (KD) downregulated matrix remodeling-related genes, while upregulated anti-tumor T-cell activity-related genes in both tumor and TDLN. CHST15 KD significantly diminished intratumoral and TDLN Ly6C/G+ myeloid-derived suppressor cells prior to TDLN T-cell expansion, suggesting that tumoral CHST15 remotely regulated myeloid-derived suppressor cell mediated T-cell suppression in the TDLN. Our findings illustrate a novel immunotherapeutic potential of tumoral CHST15 blockage by reactivating T cells in immune suppressive TDLN of PDAC.

AMPK activator AICAR in combination with anti-mouse IL10 mAb restores the functionality of intra-tumoral Tfh cells in the 4T1 mouse model.

4T1 cell-mediated TNBC breast cell carcinoma is a highly malignant mice tumor model which resembles an advanced stage of breast cancer in humans. Tumor progression occurs depending on the intra-tumoral balance of pro- and anti- tumorigenic immune cells. Enhancement of T-cell-mediated anti-tumor immunity will be advantageous for inhibiting tumor progression and improving the efficacy of cancer therapy. This study is focused on alleviating suppressed anti-tumor immune response by improving CD4+ T follicular helper cell (Tfh) response in 4T1 mice. We employed anti-IL10 mAb along with metabolic drugs 2-deoxy-D-glucose (2DG) which inhibits the glycolytic pathway and Cpt1a inhibitor Etomoxir which inhibits FAO. AMPK activator AICAR with or without anti-IL10 mAb was also used to ameliorate metabolic stress and exhaustion faced by immune cells. Our results demonstrate that synergistic treatment with 2DG/Etomoxir + anti-IL10 mAb induced Tfh cell, memory B, and GC B cell response more potently compared to treatment with 2DG or Etomoxir treatment alone as observed in several LNs and tumor tissue of 4T1 mouse. However, AICAR + anti-IL10 mAb increased the frequency of intratumoral Tfh cells, simultaneously downregulated Tfr cells; and improved humoral response by stimulating upregulation of memory B, GC B, and plasmablasts in tumor-draining, axillary, and mesenteric LNs of 4T1 mouse.

Quercetin-ferrum nanoparticles enhance photothermal therapy by modulating the tumor immunosuppressive microenvironment.

Photothermal therapy (PTT) was reported to induce synergistic immunogenic cell death (ICD) which may convert tumor cells into "therapeutic vaccines". However, this is often insufficient to prevent tumor recurrence, in part because of the immunosuppressive microenvironment in tumors. Therefore, remodeling tumor microenvironment is of great importance to enhance the therapeutic efficacy of PTT. We herein fabricated a versatile nano-photosensitizer by assembling quercetin and Ferrum ion (QFN). The released quercetin from QFN could reduce programmed death ligand 1 (PD-L1) in tumor cells by inhibiting the phosphorylation of JAK2 and STAT3, and reshape extracellular matrix (ECM) by down-regulating α-SMA+ fibroblast in tumors. Moreover, QFN could capture tumor antigen and deliver it to the tumor-draining lymph nodes after PTT, which further enhanced the activation of antigen-presenting cells. As a result, QFN-based PTT eliminated melanoma and induced long-term immune memory to prevent tumor metastasis and recurrence. This study provides an effective and translationally feasible photothermic agent for photothermal/immunotherapy. STATEMENT OF SIGNIFICANCE: The efficacy of photothermal therapy (PTT) in cancer treatment is often limited by the immunosuppressive microenvironment in tumors. Herein, we prepared a versatile photosensitizer by assembling quercetin and Ferrum ion (QFN). Upon near-infrared light irradiation, QFN-PTT induced cancer cells destruction and tumor antigen release. QFN then captured antigen and delivered it to the tumor-draining lymph nodes, thus promoting dendritic cell maturation and T cells activation. Quercetin released from QFN in tumors improved T cells infiltration and activation in tumor by regulating immunosuppressive microenvironment. The QFN-PTT-treated mice exhibited significantly elongated survival time, and gained strong anti-tumor immune memory to prevent tumor metastasis and recurrence. Thus, this work provided a simple and versatile photothermic agent, and it has important implications for designing effective and translationally feasible photosensitizers for PTT.

Expression of Interleukin-21 and Interleukin-21 receptor in lymphocytes derived from tumor-draining lymph nodes of breast cancer.

BACKGROUND: Interleukin-21 (IL-21) is produced by various cell types inducing positive and negative effects in immunity against tumors. OBJECTIVE: To investigate the expression of IL-21 by CD4+T and IL-21 receptor (IL-21R) by B lymphocytes isolated from breast-tumor draining lymph nodes (TDLNs). METHODS: Fresh lymph node samples were obtained from 45 patients with breast cancer. To assess IL-21 expression, mononuclear cells were briefly stimulated whereas IL-21R expression was assessed in unstimulated B cells. Cells were stained with antibodies for CD4, IL-21, CD19 and IL-21R and acquired by flow cytometry. RESULTS: The frequency of IL-21+CD4+T cells did not show significant association with disease parameters. However, the geometric mean fluorescence intensity (gMFI) of IL-21 in CD4+T cells was significantly lower in patients with grade III tumor than grade I + II (P = 0.042). In non-involved LNs, the intensity of IL-21 was significantly higher in patients with stage II compared with stage III (P = 0.038) and correlated negatively with the number of involved LNs. The frequency of IL-21R+CD19+B cells was significantly higher in grade III than grade I + II (P = 0.037). CONCLUSION: The higher intensity of IL-21 in CD4+T cells showed association with good prognosticators in breast cancer and warrants further investigation of the role played by IL-21 in immunity against breast cancer.

Pivotal roles of tumor-draining lymph nodes in the abscopal effects from combined immunotherapy and radiotherapy.

BACKGROUND: Currently, due to synergy enhancement of anti-tumor effects and potent stimulation of abscopal effects, combination therapy with irradiation and programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) immune checkpoint inhibition (immuno-radiotherapy, iRT) has revolutionized the therapeutic guidelines. It has been demonstrated that tumor-draining lymph nodes (TDLN) are essential for effective antitumor immunity induced by radiotherapy, immunotherapy, or iRT. Given that the function of TDLN in iRT remains unclear, this study aimed to investigate the function and mechanism of TDLN in iRT-induced abscopal effects. METHODS: The function of TDLN was evaluated using unilateral or bilateral MC38 and B16F10 subcutaneous tumor models with or without indicated TDLN. The flow cytometry, multiple immunofluorescence analysis, and NanoString analysis were utilized to detect the composition and function of the immune cells in the primary and abscopal tumor microenvironment. Additionally, we tempted to interrogate the possible mechanisms via RNA-sequencing of tumor-infiltrating lymphocytes and TDLN. RESULTS: TDLN deficiency impaired the control of tumor growth by monotherapy. Bilateral TDLN removal rather than unilateral TDLN removal substantially curtailed iRT-stimulated anti-tumor and abscopal effects. Furthermore, in the absence of TDLN, the infiltration of CD45+ and CD8+ T cells was substantially reduced in both primary and abscopal tumors, and the anti-tumor function of CD8+ T cells was attenuated as well. Additionally, the polarization of tumor-associated macrophages in primary and abscopal tumors were found to be dependent on intact bilateral TDLN. RNA-sequencing data indicated that impaired infiltration and anti-tumor effects of immune cells partially attributed to the altered secretion of components from the tumor microenvironment. CONCLUSIONS: TDLN play a critical role in iRT by promoting the infiltration of CD8+ T cells and maintaining the M1/M2 macrophage ratio.