Feasibility and Efficacy of Sentinel Lymph Node Mapping in Gastric Cancer.

BACKGROUND: Lymph node metastasis is a critical prognostic factor for patients with gastric carcinoma (GC). Sentinel lymph node (SLN) mapping has the potential to identify the initial site of draining lymph node metastasis and reduce the extent of surgical lymphadenectomy. This study aimed to evaluate the diagnostic accuracy of SLN mapping in GC. METHODS: The study enrolled 129 GC patients undergoing total or partial gastrectomy with D2 lymphadenectomy and indocyanine green fluorescence-guided SLN mapping. The primary outcomes were the negative predictive value (NPV) and sensitivity of SLN mapping. The secondary outcomes were clinicopathologic factors associated with SLN mapping accuracy and successful SLN mapping. RESULTS: The SLN detection rate in this study was 86.8 %. The study had an overall NPV of 83.1 % and an overall sensitivity of 65.8 %. The NPV was found to be significantly higher in the patients with no lymphovascular invasion (LVI) than in those with LVI (96.0 % vs 59.3 %; p < 0.001) and in the patients whose pathologic T (pT) stage lower than 3 than in those whose T stage was 3 or higher (92.0 % vs 66.7 %; p = 0.009). The sensitivity of SLN mapping was 50 % in the patients with no LVI and 33 % in the patients with a pT stage lower than 3. CONCLUSION: The study results showed that for patients with early-stage GC with no LVI, negative SLN findings may represent a potential additive predictor indicating the absence of regional LN metastasis. However, given the low sensitivity rates noted, further research is needed to identify specific patient populations that may benefit from SLN mapping in GC.

Anthocyanidin-Loaded Superabsorbent Self-Pumping Dressings for Macrophage Immunomodulation and Diabetic Wound Healing.

Management of diabetic chronic wound exudate is a serious challenge in healthcare worldwide since it is related to the speed of diabetic wound healing. However, current foam dressings not only absorb fluid to generate swelling and compress the wound to hinder wound healing but also are very thick and less comfortable to use. Herein, a superabsorbent self-pumping ultrathin dressing is reported to accelerate diabetic wound healing by achieving superior exudate absorption and management in an ultrathin state. The self-pumping dressing is composed of a drainage layer loaded with anthocyanidin and a thermoplastic polyurethane absorbent layer embedded with superabsorbent particles. The dressing realizes the self-pumping process of unidirectional exudate draining to the absorption layer through the drainage layer without significant dressing swelling to compress the diabetic wound. The dressing is experimentally proven to unidirectionally drain excessive exudate with inflammatory factors and modulate the conversion of macrophages from M1 to M2 in diabetic wounds, thereby promoting the healing of diabetic skin ulcers faster than commercial foam dressings. Therefore, the dressing provides a new idea and novel method for accelerating diabetic skin ulcer healing.

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).

Special stent for draining the abdominal abscess respectively from colon and duodenum: A case report.

BACKGROUND: Postoperative abdominal infections are an important and heterogeneous health challenge. Many samll abdominal abscesses are resolved with antibiotics, but larger or symptomatic abscesses may require procedural management. CASE SUMMARY: A 65-year-old male patient who suffered operation for the left hepatocellular carcinoma eight months ago, came to our hospital with recurrent abdominal pain, vomit, and fever for one month. Abdominal computed tomography showed that a big low-density dumbbell-shaped mass among the liver and intestine. Colonoscopy showed a submucosal mass with a fistula at colon of liver region. Gastroscopy showed a big rupture on the submucosal mass at the descending duodenum and a fistula at the duodenal bulb. Under colonoscopy, the brown liquid and pus were drained from the mass with "special stent device". Under gastroscopy, we closed the rupture of the mass with a loop and six clips for purse stitching at the descending duodenum, and the same method as colonoscopy was used to drain the brown liquid and pus from the mass. The symptom of abdominal pain, vomit and fever were relieved after the treatment. CONCLUSION: The special stent device could be effectively for draining the abdominal abscess respectively from colon and duodenum.

High expression of PD-L1 on conventional dendritic cells in tumour-draining lymph nodes is associated with poor prognosis in oral cancer.

INTRODUCTION: Oral squamous cell carcinoma (OSCC), while common and with a favorable prognosis in early stages, presents a marked reduction in survival rate upon metastasis to lymph nodes. Early detection of lymph node metastasis via biomarkers could enhance the therapeutic strategy for OSCC. Here, we explored dendritic cells (DCs) and cytotoxic T-cells in tumour-draining lymph nodes (TDLNs) as potential biomarkers. METHOD: Dendritic cells and cytotoxic T-cells in 33 lymph nodes were analyzed with multi-parameter flow cytometry in TDLNs, regional non-TDLNs surgically excised from 12 OSCC patients, and compared to 9 lymph nodes from patients with benign conditions. RESULTS: Our results displayed a higher proportion of conventional cDC1s with immunosuppressive features in TDLN. Further, high PD-L1 expression on cDC1 in TDLNs was associated with metastasis and/or recurrent disease risk. Also, elevated levels of memory CD8+ T-cells and terminally exhausted PD-1+TCF-1-CD8+ T-cells were observed in TDLNs and non-TDLNs compared to healthy lymph nodes. CONCLUSIONS: We conclude that TDLNs contain cells that could trigger an anti-tumor adaptive response, as evidenced by activated cDC1s and progenitor-like TCF-1+ T-cells. The detection of high PDL1 expression on cDC1s was indicative of TDLN metastasis and an adverse prognosis, proposing that PD-L1 on dendritic cells in TDLN could serve as a predictive biomarker of OSCC patients with a worse prognosis.

Regulatory T-cells activated in metastatic draining lymph nodes possibly suppress cancer immunity in cancer tissues of head and neck squamous cell cancer.

Regulatory T cells (Tregs) play an important role in creating an immunosuppressive microenvironment in cancer tissues. However, the mechanisms by which Tregs are activated and suppress cancer immunity remain unclear. To elucidate these mechanisms, we performed a T cell receptor (TCR) repertoire analysis of Tregs and conventional T cells in peripheral blood, draining lymph nodes (DLNs), and cancer tissues of patients with head and neck squamous cell cancer (HNSCC). We found that the TCR repertoire was skewed in cancer tissue and metastatic DLNs (M-DLNs) compared with non-metastatic DLNs, and TCR repertoire similarities in Tregs and CD8+ T cells between M-DLNs and cancer tissue were high compared with those at other sites. These results suggest that Tregs and CD8+ T cells are activated in M-DLNs and cancer tissues by cancer antigens, such as neoantigens, and shared antigens and Tregs suppress CD8+ T cell function in a cancer antigen-specific manner in M-DLNs and cancer tissue. Moreover, M-DLNs might be a source of Tregs and CD8+ T cells recruited into the cancer tissue. Therefore, targeting Tregs in M-DLNs in an antigen-specific manner is expected to be a novel immunotherapeutic strategy for HNSCCs.

Dimethyl fumarate modulates the regulatory T cell response in the mesenteric lymph nodes of mice with experimental autoimmune encephalomyelitis.

Dimethyl fumarate (DMF, Tecfidera) is an oral drug utilized to treat relapsing-remitting multiple sclerosis (MS). DMF treatment reduces disease activity in MS. Gastrointestinal discomfort is a common adverse effect of the treatment with DMF. This study aimed to investigate the effect of DMF administration in the gut draining lymph nodes cells of C57BL6/J female mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We have demonstrated that the treatment with DMF (7.5 mg/kg) significantly reduces the severity of EAE. This reduction of the severity is accompanied by the increase of both proinflammatory and anti-inflammatory mechanisms at the beginning of the treatment. As the treatment progressed, we observed an increasing number of regulatory Foxp3 negative CD4 T cells (Tr1), and anti-inflammatory cytokines such as IL-27, as well as the reduction of PGE2 level in the mesenteric lymph nodes of mice with EAE. We provide evidence that DMF induces a gradual anti-inflammatory response in the gut draining lymph nodes, which might contribute to the reduction of both intestinal discomfort and the inflammatory response of EAE. These findings indicate that the gut is the first microenvironment of action of DMF, which may contribute to its effects of reducing disease severity in MS patients.

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.

Prognostic effect of TCF1+ CD8+ T cell and TOX+ CD8+ T cell infiltration in lung adenocarcinoma.

Recent studies have highlighted the pivotal roles of T cell transcription factors TCF-1 and TOX in modulating the immune response in cancer, with TCF-1 maintaining CD8+ T cell stemness and TOX promoting T cell exhaustion. The prognostic significance of these factors in lung adenocarcinoma (LUAD) remains a critical area of investigation. The retrospective study included 191 patients with LUAD who underwent surgery, of whom 83% were in stages II and III. These patients were divided into exploratory (n = 135) and validation (n = 56) groups based on the time of diagnosis. Multiplex fluorescence immunohistochemistry was used to examine the infiltration levels of CD8+ T cells, TCF1+ CD8+ T cells, and TOX+ CD8+ T cells. The percentage of CD8+ T cells in tumor was markedly lower than that in stroma (p < 0.05). In tumor-draining lymph nodes (TDLNs) invaded by tumor, the proportion of stem-like TCF1+ CD8+ T cells was significantly decreased (p < 0.01). Importantly, higher infiltration levels of CD8+ T cells and TCF1+ CD8+ T cells were associated with improved disease-free survival (DFS) (p = 0.009 and p = 0.006, respectively) and overall survival (OS) (p = 0.018 and p = 0.010, respectively). This study underscores the potential of TCF1+ CD8+ T cells as prognostic biomarkers in LUAD, providing insights into the tumor immune microenvironment and guiding future therapeutic strategies.

Lymph node targeting strategy using a hydrogel sustained-release system to load effector memory T cells improves the anti-tumor efficacy of anti-PD-1.

Communication between tumors and lymph nodes carries substantial significance for antitumor immunotherapy. Remodeling the immune microenvironment of tumor-draining lymph nodes (TdLN) plays a key role in enhancing the anti-tumor ability of immunotherapy. In this study, we constructed a biomimetic artificial lymph node structure composed of F127 hydrogel loading effector memory T (TEM) cells and PD-1 inhibitors (aPD-1). The biomimetic lymph nodes facilitate the delivery of TEM cells and aPD-1 to the TdLN and the tumor immune microenvironment, thus realizing effective and sustained anti-tumor immunotherapy. Exploiting their unique gel-forming and degradation properties, the cold tumors were speedily transformed into hot tumors via TEM cell supplementation. Meanwhile, the efficacy of aPD-1 was markedly elevated compared with conventional drug delivery methods. Our finding suggested that the development of F127@TEM@aPD-1 holds promising potential as a future novel clinical drug delivery technique. STATEMENT OF SIGNIFICANCE: F127@TEM@aPD-1 show unique advantages in cancer treatment. When injected subcutaneously, F127@TEM@aPD-1 can continuously supplement TEM cells and aPD-1 to tumor draining lymph nodes (TdLN) and the tumor microenvironment, not only improving the efficacy of ICB therapy through slow release, but also exhibiting dual regulatory effects on the tumor and TdLN.

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.

Natural Killer Cell Subsets in Tumor Draining Lymph Nodes of Patients with Bladder Cancer and Their Clinical Implications.

Background: Natural killer (NK) cells are crucial innate components in anti-tumor immunity. However, the clinical impacts and their phenotypes in bladder cancer (BC) remain unclear. Objective: To assess the clinical significance of NK cell subsets in tumor-draining lymph nodes of patients with BC. Methods: In a cross-sectional study, pelvic lymph nodes were obtained from 49 untreated patients with BC. Mononuclear cells were isolated and immunophenotyped using CD3, CD56, CD16, CD27, and CD11b markers. NK cells were then classified based on their expression patterns of CD56/CD16 (conventional) and CD27/CD11b (new). Results: On average, NK cells constituted 2.99±1.44% of the total lymphocytes in the draining lymph node of patients with BC. The CD56dim and regulatory NK subsets (CD27+CD11b+/-) were the predominant old and new NK, respectively. The NK cells significantly increased in patients with at least one involved node (LN+) compared with those with free nodes (LN-; p=0.022). Conversely, CD56dimCD16- subset significantly decreased in higher stages (p=0.032) and in tumors with muscle invasion (p=0.038). Significant variations were also observed in different T-stages (p<0.05). Regarding new classification, the frequency of CD11b+ regulatory NK cells was significantly lower in node-positive patients (p=0.025). Conclusion: These findings emphasize the dynamic nature of NK cell subsets in bladder cancer and their potential relevance in disease progression and management, suggesting potential implications for therapeutic strategies targeting these specific subsets.

Histopathological analysis of the wall enhancement of the spinal dural arteriovenous fistulae's draining veins.

OBJECTIVE: The mechanism behind SDAVF is still unclear. We discovered that the vessel wall of the SDAVF-DV occasionally showed enhancement in MRI, and this study assessed the relationship between the enhancement of the draining vein's wall and its histology. METHODS: For histopathologic analysis, 16 draining vein samples from 16 patients with SDAVF were included, 3 normal arteries and 3 normal veins were chosen as comparison. We assessed the imaging and microscopic characteristics of the draining veins in SDAVF patients. The former included the presence of significant enhancement of the wall of the draining vein in MRI, and the latter included the adherence, aggregation, infiltration of pro-inflammatory factors and inflammatory cells. Immuno-histochemical staining was performed using IL-1ß, IL-8, TGF-ß as well as MPO and MMP-9, and positive results were counted. Multiple logistic regression analysis was used to determine whether the infiltration of inflammatory cells was connected to vessel wall enhancement in the SDAVF-DV. RESULTS: Infiltration of inflammatory cells was significantly higher in SDAVF-DV compared to normal vessels, 7 out of 16 patients significantly had enhancement of the vessel wall of SDAVF-DV, and logistic regression analysis showed that samples with more infiltration of inflammatory cells were more likely to show enhancement of the SDAVF-DV walls. CONCLUSION: There was considerable inflammatory cells infiltration in SDAVF-DV, and this may explain why their vessel wall had such a significant enhancement in MRI.

Case report: Management of generalized infection and draining tracts of the frontomaxillary region in a dog.

Objective: This study aims to report the surgical and medical management of generalized chronic maxillofacial infection with multiple intra- and extraoral draining tracts in a dog. Case summary: A 6 years-old, male neutered pit bull terrier dog underwent a staged procedure. First, a diagnostic work-up including hematologic and biochemical analysis, conventional computed tomography (CT) with contrast of the skull, and a rhinoscopic evaluation of the draining tracts was performed. Samples were obtained for histopathological, microbial, and fungal testing. Second, a 4 week course of antimicrobials based on culture and sensitivity results was administered. Third, an extraoral approach to soft tissue reconstruction was accomplished as a first stage in the repair process. Finally, an intraoral approach to repair the oronasal fistulous draining tracts was performed. A 6 months follow-up skull CT revealed various stages of repair and remodeling and adequate soft tissue healing. Clinical relevance: A staged procedure is a suitable option to treat chronic and generalized frontal and maxillary infection with multiple intra- and extraoral fistulous draining tracts in dogs.

Methods behind oncolytic virus-based DC vaccines in cancer: Toward a multiphase combined treatment strategy for Glioblastoma (GBM) patients.

Glioblastoma (GBM) remains an orphan cancer disease with poor outcome. Novel treatment strategies are needed. Immunotherapy has several modes of action. The addition of active specific immunotherapy with dendritic cell vaccines resulted in improved overall survival of patients. Integration of DC vaccination within the first-line combined treatment became a challenge, and immunogenic cell death immunotherapy during chemotherapy was introduced. We used a retrospective analysis using real world data to evaluate the complex combined treatment, which included individualized multimodal immunotherapy during and after standard of care, and which required adaptations during treatment, and found a further improvement of overall survival. We also discuss the use of real world data as evidence. Novel strategies to move the field of individualized multimodal immunotherapy forward for GBM patients are reviewed.

Adoptive immunotherapy with cells from tumor-draining lymph nodes activated and expanded in vitro.

Tumor-draining lymph nodes (tumor-DLNs) provide a rich source of tumor-reactive lymphocytes which can be used in adoptive immunotherapy (AIT) and that circumvent the need to resect autologous tumor, without the challenges and shortcomings associated with using autologous tumor or anti-CD3 monoclonal antibody. Bryostatin/Ionomycin (Bryo/Io) provide a useful method of activating tumor-DLNs such that they can readily be expanded to sufficient numbers to be used in AIT, and growing the tumor-DLN lymphocytes in the gamma chain cytokines IL-7 plus IL-15 is superior to IL-2 in terms of T cell numbers and phenotype. AIT with these cells induces tumor regression and provides protection against metastases and future tumor challenge. Here, we provide a stepwise protocol to sensitize tumor-DLN cells in donor mice, activate tumor-DLN T cells ex vivo using Bryo/Io, expansion of these cells in gamma chain cytokines and adoptive transfer of the expanded cells back into tumor-bearing hosts. Methods relevant to these experiments, such as injecting tumor cells intravenously and monitoring for pulmonary metastases, tumor volume measurement and resection, and use of luciferase-expressing tumor cells to monitor for metastases following resection, are described in detail. The methods outlined herein can be easily adapted to suit similar experiments across multiple tumor cell lines and syngeneic mouse models.

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.

Phototruncation cell tracking with near-infrared photoimmunotherapy using heptamethine cyanine dye to visualise migratory dynamics of immune cells.

BACKGROUND: Noninvasive in vivo cell tracking is valuable in understanding the mechanisms that enhance anti-cancer immunity. We have recently developed a new method called phototruncation-assisted cell tracking (PACT), that uses photoconvertible cell tracking technology to detect in vivo cell migration. This method has the advantages of not requiring genetic engineering of cells and employing tissue-penetrant near-infrared light. METHODS: We applied PACT to monitor the migration of immune cells between a tumour and its tumour-draining lymph node (TDLN) after near-infrared photoimmunotherapy (NIR-PIT). FINDINGS: PACT showed a significant increase in the migration of dendritic cells (DCs) and macrophages from the tumour to the TDLN immediately after NIR-PIT. This migration by NIR-PIT was abrogated by inhibiting the sphingosine-1-phosphate pathway or Gαi signaling. These results were corroborated by intranodal immune cell profiles at two days post-treatment; NIR-PIT significantly induced DC maturation and increased and activated the CD8+ T cell population in the TDLN. Furthermore, PACT revealed that NIR-PIT significantly enhanced the migration of CD8+ T cells from the TDLN to the tumour four days post-treatment, which was consistent with the immunohistochemical assessment of tumour-infiltrating lymphocytes and tumour regression. INTERPRETATION: Immune cells dramatically migrated between the tumour and TDLN following NIR-PIT, indicating its potential as an immune-stimulating therapy. Also, PACT is potentially applicable to a wide range of immunological research. FUNDING: This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Centre for Cancer Research (grant number: ZIA BC011513 and ZIA BC011506).

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.

NIR-II Ratiometric Fluorescence Probes Enable Precise Determination of the Metastatic Status of Sentinel Lymph Nodes.

Accurately determining the metastatic status of sentinel lymph nodes (SLNs) through noninvasive imaging with high imaging resolution and sensitivity is crucial for cancer therapy. Herein, we report a dual-tracer-based NIR-II ratiometric fluorescence nanoplatform combining targeted and nontargeted moieties to determine the metastatic status of SLNs through the recording of ratio signals. Ratiometric fluorescence imaging revealed approximately 2-fold increases in signals in tumor-draining SLNs compared to inflamed and normal SLNs. Additionally, inflamed SLNs were diagnosed by combining the ratio value with the enlarged size outputted by NIR-II fluorescence imaging. The metastatic status diagnostic results obtained through NIR-II ratiometric fluorescence signals were further confirmed by standard H&E staining, indicating that the ratiometric fluorescence strategy could achieve distant metastases detection. Furthermore, the superior imaging quality of ratiometric probes enables visualization of the detailed change in the lymphatic network accompanying tumor growth. Compared to clinically available and state-of-the-art NIR contrast agents, our dual-tracer-based NIR-II ratiometric fluorescence probes provide significantly improved performance, allowing for the quick assessment of lymphatic function and guiding the removal of tumor-infiltrating SLNs during cancer surgery.