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BackgroundMetastatic colorectal cancer (mCRC) is a common and highly lethal gastrointestinal malignancy. Although chemotherapy combined with bevacizumab is a standard first-line treatment, post-treatment resistance severely limits patients’ long-term survival. While the remodeling of tumor-associated macrophages (TAMs) is closely related to targeted therapy resistance, the specific impact of TAMs and their programmed death-ligand 1 (PD-L1) expression on post-treatment resistance and prognosis remains unclear. This study aimed to investigate the spatial distribution and immunophenotypic characteristics of CD68+CD163+ M2-like TAMs and their association with post-treatment resistance and survival.MethodsWe retrospectively analyzed clinical data and tumor tissues from 44 patients with mCRC who received first-line chemotherapy plus bevacizumab and were categorized into resistant and non-resistant groups. Multiplex immunofluorescence and digital image-based quantitative analysis were used to evaluate macrophage markers (CD68 and CD163) and PD-L1 expression in the whole tumor section, tumor areas, and stromal areas.ResultsCompared with the non-resistant group, resistant patients showed significantly increased proportions and densities of CD68+CD163+ M2-like TAMs and CD68+CD163+PD-L1+ M2-like TAMs in the tumor areas (all p < 0.05). The infiltration levels of these macrophage subsets were significantly associated with post-treatment resistance, but not with conventional clinicopathological features such as sex, age, or tumor stage. Survival analysis showed that a high density of intratumoral CD68+CD163+ M2-like TAMs was significantly associated with shorter overall survival (OS) (p < 0.05) and showed a trend toward shorter progression-free survival (PFS) (p = 0.06). Multivariate Cox regression further demonstrated that dense intratumoral infiltration of CD68+CD163+ M2-like TAMs was independently associated with unfavorable OS and PFS.ConclusionIn mCRC patients exhibiting resistance to chemotherapy combined with bevacizumab, the infiltration of intratumoral CD68+CD163+ M2-like TAMs and their PD-L1-expressing subsets is significantly elevated. Notably, a high density of intratumoral CD68+CD163+ M2-like TAMs serves as an independent poor prognostic marker for both PFS and OS. These findings demonstrate that the spatial enrichment of CD68+CD163+ M2-like TAMs within the tumor microenvironment is closely associated with treatment resistance, suggesting that macrophage-targeted interventions may represent a potential strategy to enhance the efficacy of anti-tumor therapies in mCRC.

In the development of a multiplex immunofluorescence (IF) platform and the optimization and validation of new multiplex IF panels using a tyramide signal amplification system, several technical requirements are important for high-quality staining, analysis, and results. The aim of this review is to discuss the basic requirements for performing multiplex IF tyramide signal amplification (TSA) in formalin-fixed, paraffin-embedded cancer tissues to support translational oncology research. Our laboratory has stained approximately 4000 formalin-fixed, paraffin-embedded tumor samples using the multiplex IF TSA system for immune profiling of several labeled biomarkers in a single slide to elucidate cancer biology at a protein level and identify therapeutic targets and biomarkers. By analyzing several proteins in thousands of cells on a single slide, this technique provides a systems-level view of various processes in various tumor tissues. Although this technology shows high flexibility in cancer studies, it presents several challenges when applied to study different histology cancers. Our experience shows that adequate antibody validation, staining optimization, analysis strategies, and data generation are important steps for generating quality results. Tissue management, fixation procedures, storage, and cutting can also affect the results of the assay and must be standardized. Overall, this method is reliable for supporting translational research given a precise, step-by-step approach.

Optimisation and validation of a multiplex immunofluorescence (mIF) workflow, from staining to digital image analysis (DIA), ensure assay robustness. Chromogenic immunohistochemistry (IHC) and fluorescent singleplexes are fundamental in this process, particularly when biomarkers are co‐expressed. We describe our experience developing two mIF panels and the various parameters of staining, scanning and DIA to consider when standardising a digital pathology workflow. Multiplex immunofluorescence is a powerful tool for the simultaneous detection of tissue‐based biomarkers, revolutionising traditional immunohistochemistry. The Opal methodology allows up to eight biomarkers to be measured concomitantly without cross‐reactivity, permitting identification of different cell populations within the tumour microenvironment. In this study, we aimed to validate a multiplex immunofluorescence workflow in two complementary multiplex panels and evaluate the tumour immune microenvironment in colorectal cancer (CRC) formalin‐fixed paraffin‐embedded tissue. We stained CRC and tonsil samples using Opal multiplex immunofluorescence on a Leica BOND RX immunostainer. We then acquired images on an Akoya Vectra Polaris and performed multispectral unmixing using inform. Antibody panels were validated on tissue microarray sections containing cores from six normal tissue types, using qupath for image analysis. Comparisons between chromogenic immunohistochemistry and multiplex immunofluorescence on consecutive sections from the same tissue microarray showed significant correlation (rs > 0.9, P‐value < 0.0001), validating both panels. We identified many factors that influenced the quality of the acquired fluorescent images, including biomarker co‐expression, staining order, Opal‐antibody pairing, sample thickness, multispectral unmixing and biomarker detection order during image analysis. Overall, we report the optimisation and validation of a multiplex immunofluorescence process, from staining to image analysis, ensuring assay robustness. Our multiplex immunofluorescence protocols permit the accurate detection of multiple immune markers in various tissue types, using a workflow that enables rapid processing of samples, above and beyond previous workflows.

Background Fibrinogen-like protein 1 (FGL1)—Lymphocyte activating gene 3 (LAG-3) pathway is a promising immunotherapeutic target and has synergistic effect with programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1). However, the prognostic significance of FGL1-LAG-3 pathway and the correlation with PD-L1 in hepatocellular carcinoma (HCC) remain unknown. Methods The levels of LAG-3, FGL1, PD-L1 and cytotoxic T (CD8 + T) cells in 143 HCC patients were assessed by multiplex immunofluorescence. Associations between the marker’s expression and clinical significances were studied. Results We found FGL1 and LAG-3 densities were elevated while PD-L1 and CD8 were decreased in HCC tissues compared to adjacent normal liver tissues. High levels of FGL1 were strongly associated with high densities of LAG-3 + cells but not PD-L1. CD8 + T cells densities had positive correlation with PD-L1 levels and negative association with FGL1 expression. Elevated densities of LAG-3 + cells and low levels of CD8 + T cells were correlated with poor disease outcome. Moreover, LAG-3 + cells deteriorated patient stratification based on the abundance of CD8 + T cells. Patients with positive PD-L1 expression on tumor cells (PD-L1 TC + ) tended to have an improved survival than that with negative PD-L1 expression on tumor cells (PD-L1 TC − ). Furthermore, PD-L1 TC − in combination with high densities of LAG-3 + cells showed the worst prognosis, and PD-L1 TC + patients with low densities of LAG-3 + cells had the best prognosis. Conclusions LAG-3, FGL1, PD-L1 and CD8 have distinct tissue distribution and relationships with each other. High levels of LAG-3 + cells and CD8 + T cells represent unfavorable and favorable prognostic biomarkers for HCC respectively.

[This corrects the article DOI: 10.3389/fimmu.2023.1324618.].

Background Neoadjuvant chemoimmunotherapy treatment (NCIT) has achieved great success for non-small cell lung cancer (NSCLC); however, the intrinsic mechanism underlying this treatment remains unclear. Methods Thirty-two patients with stage IIA–IIIC NSCLC who underwent surgery after NCIT were included in this retrospective study. Multiplex immunofluorescence (mIF) staining and image analysis assays were performed on the samples collected before and after NCIT for each patient. RNA analyses was applied to confirm the mIF results. Results Among the enrolled patients, 14 achieved major pathological response or pathological complete response (pCR) and were defined as the ‘response’ group, whereas 18 patients did not respond well to NCIT and were defined as the ‘nonresponse’ group. The results of the mIF assays revealed an overall increase in tumor immune lymphocytes (TILs) after NCIT in the stroma area ( p  = 0.03) rather than the tumor area ( p  = 0.86). The percentage of CD8+ T cells and tertiary lymphoid structure counts in both the response and nonresponse groups increased significantly after NCIT compared with before NCIT. CD3+ T cells and FOXP3+ cells decreased significantly in the response group but remained unchanged or increased in the nonresponse group. A comparison of the response and nonresponse groups showed that CD3, FOXP3+ and CD8+/PD-1+ cells before NCIT may serve as predictors of the response to neoadjuvant immunotherapy. The RNA analyses confirmed the mIF results that TILs were elevated after NCIT. Conclusions The infiltration of immune cells before NCIT was correlated with pathologic complete response, which enhanced the TILs as a promising predictor for selecting patients who were more likely to benefit from NCIT.

Background Immune checkpoint inhibitors (ICI) are approved for treatment of recurrent or metastatic oropharyngeal head and neck squamous cell carcinoma in the first- and second-line settings. However, only 15–20% of patients benefit from this treatment, a feature increasingly ascribed to the peculiar characteristics of the tumor immune microenvironment (TIME). Methods Immune-related gene expression profiling (GEP) and multiplex immunofluorescence (mIF) including spatial proximity analysis, were used to characterize the TIME of 39 treatment-naïve oropharyngeal squamous cell carcinomas (OPSCC) and the corresponding lymph node metastases. GEP and mIF results were correlated with disease-free survival (DFS). HPV-positive tumors disclosed a stronger activation of several immune signalling pathways, as well as a higher expression of genes related to total tumor-infiltrating lymphocytes, CD8 T cells, cytotoxic cells and exhausted CD8 cells, than HPV-negative patients. Accordingly, mIF revealed that HPV-positive lesions were heavily infiltrated as compared to HPV-negative counterparts, with a higher density of T cells and checkpoint molecules. CD8+ T cells appeared in closer proximity to tumor cells, CD163+ macrophages and FoxP3+ cells in HPV-positive primary tumors, and related metastases. In HPV-positive lesions, PD-L1 expression was increased as compared to HPV-negative samples, and PD-L1+ tumor cells and macrophages were closer to PD-1+ cytotoxic T lymphocytes. Considering the whole cohort, a positive correlation was observed between DFS and higher levels of activating immune signatures and T cell responses, higher density of PD-1+ T cells and their closer proximity to tumor cells or PD-L1+ macrophages. HPV-positive patients with higher infiltration of T cells and macrophages had a longer DFS, while CD163+ macrophages had a negative role in prognosis of HPV-negative patients. Conclusions Our results suggest that checkpoint expression may reflect an ongoing antitumor immune response. Thus, these observations provide the rationale for the incorporation of ICI in the loco-regional therapy strategies for patients with heavily infiltrated treatment-naïve OPSCC, and for the combination of ICI with tumor-specific T cell response inducers or TAM modulators for the “cold” OPSCC counterparts.

Background Anti-PD-(L)1 immunotherapy has been recommended for non-small cell lung cancer (NSCLC) patients with lymph node metastases (LNM). However, the exact functional feature and spatial architecture of tumor-infiltrating CD8 + T cells remain unclear in these patients. Methods Tissue microarrays (TMAs) from 279 IA-IIIB NSCLC samples were stained by multiplex immunofluorescence (mIF) for 11 markers (CD8, CD103, PD-1, Tim3, GZMB, CD4, Foxp3, CD31, αSMA, Hif-1α, pan-CK). We evaluated the density of CD8 + T-cell functional subsets, the mean nearest neighbor distance (mNND) between CD8 + T cells and neighboring cells, and the cancer-cell proximity score (CCPS) in invasive margin (IM) as well as tumor center (TC) to investigate their relationships with LNM and prognosis. Results The densities of CD8 + T-cell functional subsets, including predysfunctional CD8 + T cells (T predys ) and dysfunctional CD8 + T cells (T dys ), in IM predominated over those in TC ( P  < 0.001). Multivariate analysis identified that the densities of CD8 + T predys cells in TC and CD8 + T dys cells in IM were significantly associated with LNM [OR = 0.51, 95%CI (0.29–0.88), P  = 0.015; OR = 5.80, 95%CI (3.19–10.54), P  < 0.001; respectively] and recurrence-free survival (RFS) [HR = 0.55, 95%CI (0.34–0.89), P  = 0.014; HR = 2.49, 95%CI (1.60–4.13), P  = 0.012; respectively], independent of clinicopathological factors. Additionally, shorter mNND between CD8 + T cells and their neighboring immunoregulatory cells indicated a stronger interplay network in the microenvironment of NSCLC patients with LNM and was associated with worse prognosis. Furthermore, analysis of CCPS suggested that cancer microvessels (CMVs) and cancer-associated fibroblasts (CAFs) selectively hindered CD8 + T cells from contacting with cancer cells, and were associated with the dysfunction of CD8 + T cells. Conclusion Tumor-infiltrating CD8 + T cells were in a more dysfunctional status and in a more immunosuppressive microenvironment in patients with LNM compared with those without LNM.

Background Despite major improvements in treatment of HER2-positive metastatic breast cancer (MBC), only few patients achieve complete remission and remain progression free for a prolonged time. The tumor immune microenvironment plays an important role in the response to treatment in HER2-positive breast cancer and could contain valuable prognostic information. Detailed information on the cancer-immune cell interactions in HER2-positive MBC is however still lacking. By characterizing the tumor immune microenvironment in patients with HER2-positive MBC, we aimed to get a better understanding why overall survival (OS) differs so widely and which alternative treatment approaches may improve outcome. Methods We included all patients with HER2-positive MBC who were treated with trastuzumab-based palliative therapy in the Netherlands Cancer Institute between 2000 and 2014 and for whom pre-treatment tissue from the primary tumor or from metastases was available. Infiltrating immune cells and their spatial relationships to one another and to tumor cells were characterized by immunohistochemistry and multiplex immunofluorescence. We also evaluated immune signatures and other key pathways using next-generation RNA-sequencing data. With nine years median follow-up from initial diagnosis of MBC, we investigated the association between tumor and immune characteristics and outcome. Results A total of 124 patients with 147 samples were included and evaluated. The different technologies showed high correlations between each other. T-cells were less prevalent in metastases compared to primary tumors, whereas B-cells and regulatory T-cells (Tregs) were comparable between primary tumors and metastases. Stromal tumor-infiltrating lymphocytes in general were not associated with OS. The infiltration of B-cells and Tregs in the primary tumor was associated with unfavorable OS. Four signatures classifying the extracellular matrix of primary tumors showed differential survival in the population as a whole. Conclusions In a real-world cohort of 124 patients with HER2-positive MBC, B-cells, and Tregs in primary tumors are associated with unfavorable survival. With this paper, we provide a comprehensive insight in the tumor immune microenvironment that could guide further research into development of novel immunomodulatory strategies. Graphical Abstract

This study attempted to profile the tumor immune microenvironment (TIME) of non-small cell lung cancer (NSCLC) by multiplex immunofluorescence of 681 NSCLC cases. The number, density, and proportion of 26 types of immune cells in tumor nest and tumor stroma were evaluated, revealing some close interactions particularly between intrastromal neutrophils and intratumoral regulatory T cells (Treg) ( r 2 = 0.439, P < 0.001), intrastromal CD4+CD38+ T cells and CD20-positive B cells ( r 2 = 0.539, P < 0.001), and intratumoral CD8-positive T cells and M2 macrophages expressing PD-L1 ( r 2  = 0.339, P < 0.001). Three immune subtypes correlated with distinct immune characteristics were identified using the unsupervised consensus clustering approach. The immune-activated subtype had the longest disease-free survival (DFS) and demonstrated the highest infiltration of CD4-positive T cells, CD8-positive T cells, and CD20-positive B cells. The immune-defected subtype was rich in cancer stem cells and macrophages, and these patients had the worst prognosis. The immune-exempted subtype had the highest levels of neutrophils and Tregs. Intratumoral CD68-positive macrophages, M1 macrophages, and intrastromal CD4+ cells, CD4+FOXP3- cells, CD8+ cells, and PD-L1+ cells were further found to be the most robust prognostic biomarkers for DFS, which were used to construct and validate the immune-related risk score for risk stratification (high vs . median vs . low) and the prediction of 5-year DFS rates (23.2% vs . 37.9% vs . 43.1%, P < 0.001). In conclusion, the intricate and intrinsic structure of TIME in NSCLC was demonstrated, showing potency in subtyping and prognostication.