Multiplexed analysis of macrophage polarisation in pulmonary metastases of microsatellite stable colorectal cancer.

Tumour-associated macrophages (TAMs) express a continuum of phenotypes ranging from an anti-tumoural M1-like phenotype to a pro-tumoural M2-like phenotype. During cancer progression, TAMs may shift to a more M2-like polarisation state, but the role of TAMs in CRC metastases is unclear. We conducted a comprehensive spatial and prognostic analysis of TAMs in CRC pulmonary metastases and corresponding primary tumours using multiplexed immunohistochemistry and machine learning-based image analysis. We obtained data from 106 resected pulmonary metastases and 74 corresponding primary tumours. TAMs in the resected pulmonary metastases were located closer to the cancer cells and presented a more M2-like polarised state in comparison to the primary tumours. Higher stromal M2-like macrophage densities in the invasive margin of pulmonary metastases were associated with worse 5-year overall survival (HR 3.19, 95% CI 1.35-7.55, p = 0.008). The results of this study highlight the value of multiplexed analysis of macrophage polarisation in cancer metastases and might have clinical implications in future cancer therapy.

Quantitative Multiplexed Analysis of Indoleamine 2,3-Dioxygenase (IDO) and Arginase-1 (ARG1) Expression and Myeloid Cell Infiltration in Colorectal Cancer.

Indoleamine 2,3-dioxygenase (IDO) and arginase-1 (ARG1) are amino acid-metabolizing enzymes, frequently highly expressed in cancer. Their expression may deplete essential amino acids, lead to immunosuppression, and promote cancer growth. Still, their expression patterns, prognostic significance, and spatial localization in the colorectal cancer microenvironment are incompletely understood. Using a custom 10-plex immunohistochemistry assay and supervised machine learning-based digital image analysis, we characterized IDO and ARG1 expression in monocytic cells, granulocytes, mast cells, and tumor cells in 833 colorectal cancer patients. We evaluated the prognostic value and spatial arrangement of IDO- and ARG1-expressing myeloid and tumor cells. IDO was mainly expressed not only by monocytic cells but also by some tumor cells, whereas ARG1 was predominantly expressed by granulocytes. Higher density of IDO+ monocytic cells was an independent prognostic factor for improved cancer-specific survival both in the tumor center (Ptrend = .0002; hazard ratio [HR] for the highest ordinal category Q4 [vs Q1], 0.51; 95% CI, 0.33-0.79) and the invasive margin (Ptrend = .0015). Higher density of granulocytes was associated with prolonged cancer-specific survival in univariable models, and higher FCGR3+ARG1+ neutrophil density in the tumor center also in multivariable analysis (Ptrend = .0020). Granulocytes were, on average, located closer to tumor cells than monocytic cells. Furthermore, IDO+ monocytic cells and ARG1- granulocytes were closer than IDO- monocytic cells and ARG1+ granulocytes, respectively. The mRNA expression of the IDO1 gene was assessed in myeloid and tumor cells using publicly available single-cell RNA sequencing data for 62 colorectal cancers. IDO1 was mainly expressed in monocytes and dendritic cells, and high IDO1 activity in monocytes was associated with enriched immunostimulatory pathways. Our findings provided in-depth information about the infiltration patterns and prognostic value of cells expressing IDO and/or ARG1 in the colorectal cancer microenvironment, highlighting the significance of host immune response in tumor progression.

Bayesian risk prediction model for colorectal cancer mortality through integration of clinicopathologic and genomic data.

Routine tumor-node-metastasis (TNM) staging of colorectal cancer is imperfect in predicting survival due to tumor pathobiological heterogeneity and imprecise assessment of tumor spread. We leveraged Bayesian additive regression trees (BART), a statistical learning technique, to comprehensively analyze patient-specific tumor characteristics for the improvement of prognostic prediction. Of 75 clinicopathologic, immune, microbial, and genomic variables in 815 stage II-III patients within two U.S.-wide prospective cohort studies, the BART risk model identified seven stable survival predictors. Risk stratifications (low risk, intermediate risk, and high risk) based on model-predicted survival were statistically significant (hazard ratios 0.19-0.45, vs. higher risk; P < 0.0001) and could be externally validated using The Cancer Genome Atlas (TCGA) data (P = 0.0004). BART demonstrated model flexibility, interpretability, and comparable or superior performance to other machine-learning models. Integrated bioinformatic analyses using BART with tumor-specific factors can robustly stratify colorectal cancer patients into prognostic groups and be readily applied to clinical oncology practice.

Immunological and prognostic significance of tumour necrosis in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) causes the second most cancer deaths worldwide, but the disease course varies according to tumour characteristics and immunological factors. Our objective was to examine the associations of tumour necrosis with tumour characteristics, immune cell infiltrates, serum cytokine concentrations, as well as prognosis in CRC. METHODS: Three independent CRC cohorts, including 1413 patients, were analysed. Associations of the areal percentage of tumour necrosis with clinicopathologic parameters, tumour infiltrating immune cells, cytokine concentrations in systemic and mesenteric vein blood, and survival were examined. RESULTS: Higher tumour necrosis percentage associated with shorter colorectal cancer-specific survival independent of tumour grade, T, N or M-class, mismatch repair status, BRAF status, and other possible confounding factors. In the largest cohort (N = 1100), the HR for high tumour necrosis percentage (≥40% vs. <3%) was 3.22 (95% CI 1.68-6.17, Ptrend < 0.0001). Tumour necrosis percentage positively correlated with peripheral serum levels of CXCL8, a proinflammatory chemokine, and negatively correlated with mesenteric serum levels of CXCL10 and mast cell densities in the invasive margin of the tumour. CONCLUSIONS: Our results support the value of tumour necrosis as a prognostic factor in colorectal cancer. CXCL8 may have a role in the systemic effects of tumour necrosis.

A pan-cancer analysis shows immunoevasive characteristics in NRF2 hyperactive squamous malignancies.

The NRF2 pathway is frequently activated in various cancer types, yet a comprehensive analysis of its effects across different malignancies is currently lacking. We developed a NRF2 activity metric and utilized it to conduct a pan-cancer analysis of oncogenic NRF2 signaling. We identified an immunoevasive phenotype where high NRF2 activity is associated with low interferon-gamma (IFNγ), HLA-I expression and T cell and macrophage infiltration in squamous malignancies of the lung, head and neck area, cervix and esophagus. Squamous NRF2 overactive tumors comprise a molecular phenotype with SOX2/TP63 amplification, TP53 mutation and CDKN2A loss. These immune cold NRF2 hyperactive diseases are associated with upregulation of immunomodulatory NAMPT, WNT5A, SPP1, SLC7A11, SLC2A1 and PD-L1. Based on our functional genomics analyses, these genes represent candidate NRF2 targets, suggesting direct modulation of the tumor immune milieu. Single-cell mRNA data shows that cancer cells of this subtype exhibit decreased expression of IFNγ responsive ligands, and increased expression of immunosuppressive ligands NAMPT, SPP1 and WNT5A that mediate signaling in intercellular crosstalk. In addition, we discovered that the negative relationship of NRF2 and immune cells are explained by stromal populations of lung squamous cell carcinoma, and this effect spans multiple squamous malignancies based on our molecular subtyping and deconvolution data.

Spatially resolved multimarker evaluation of CD274 (PD-L1)/PDCD1 (PD-1) immune checkpoint expression and macrophage polarisation in colorectal cancer.

BACKGROUND: The CD274 (PD-L1)/PDCD1 (PD-1) immune checkpoint interaction may promote cancer progression, but the expression patterns and prognostic significance of PD-L1 and PD-1 in the colorectal cancer microenvironment are inadequately characterised. METHODS: We used a custom 9-plex immunohistochemistry assay to quantify the expression patterns of PD-L1 and PD-1 in macrophages, T cells, and tumour cells in 910 colorectal cancer patients. We evaluated cancer-specific mortality according to immune cell subset densities using multivariable Cox regression models. RESULTS: Compared to PD-L1- macrophages, PD-L1+ macrophages were more likely M1-polarised than M2-polarised and located closer to tumour cells. PD-L1+ macrophage density in the invasive margin associated with longer cancer-specific survival [Ptrend = 0.0004, HR for the highest vs. lowest quartile, 0.52; 95% CI: 0.34-0.78]. T cell densities associated with longer cancer-specific survival regardless of PD-1 expression (Ptrend < 0.005 for both PD-1+ and PD-1- subsets). Higher densities of PD-1+ T cell/PD-L1+ macrophage clusters associated with longer cancer-specific survival (Ptrend < 0.005). CONCLUSIONS: PD-L1+ macrophages show distinct polarisation profiles (more M1-like), spatial features (greater co-localisation with tumour cells and PD-1+ T cells), and associations with favourable clinical outcome. Our comprehensive multimarker assessment could enhance the understanding of immune checkpoints in the tumour microenvironment and promote the development of improved immunotherapies.

Spatially Resolved Single-Cell Assessment of Pancreatic Cancer Expression Subtypes Reveals Co-expressor Phenotypes and Extensive Intratumoral Heterogeneity.

Pancreatic ductal adenocarcinoma (PDAC) has been classified into classical and basal-like transcriptional subtypes by bulk RNA measurements. However, recent work has uncovered greater complexity to transcriptional subtypes than was initially appreciated using bulk RNA expression profiling. To provide a deeper understanding of PDAC subtypes, we developed a multiplex immunofluorescence (mIF) pipeline that quantifies protein expression of six PDAC subtype markers (CLDN18.2, TFF1, GATA6, KRT17, KRT5, and S100A2) and permits spatially resolved, single-cell interrogation of pancreatic tumors from resection specimens and core needle biopsies. Both primary and metastatic tumors displayed striking intratumoral subtype heterogeneity that was associated with patient outcomes, existed at the scale of individual glands, and was significantly reduced in patient-derived organoid cultures. Tumor cells co-expressing classical and basal markers were present in > 90% of tumors, existed on a basal-classical polarization continuum, and were enriched in tumors containing a greater admixture of basal and classical cell populations. Cell-cell neighbor analyses within tumor glands further suggested that co-expressor cells may represent an intermediate state between expression subtype poles. The extensive intratumoral heterogeneity identified through this clinically applicable mIF pipeline may inform prognosis and treatment selection for patients with PDAC. SIGNIFICANCE: A high-throughput pipeline using multiplex immunofluorescence in pancreatic cancer reveals striking expression subtype intratumoral heterogeneity with implications for therapy selection and identifies co-expressor cells that may serve as intermediates during subtype switching.

TLR2 and TLR4 in colorectal cancer: relationship to tumor necrosis and markers of systemic inflammation.

In colorectal cancer (CRC), systemic inflammation is associated with poor prognosis, but the underlying mechanisms are not fully characterized. Tumor necrosis may contribute to systemic inflammation by inducing interleukin (IL)-6 signaling, and proinflammatory cytokines such as IL-6 and IL-8, and matrix metalloproteinase (MMP)-8 also are linked to adverse CRC outcomes. Because Toll-like receptors (TLRs) are important mediators of inflammatory responses, we investigated the roles of TLR2 and TLR4 in CRC-associated systemic inflammatory responses, especially tumor necrosis. In 118 patients with CRC, extensive tumor necrosis was associated with low TLR4 expression in tumor cells. Tumor cell TLR4 expression was inversely correlated with serum IL-6 and MMP-8 levels, blood total leukocyte and neutrophil counts, and serum C-reactive protein levels. Tumor cell TLR2 expression was not significantly associated with necrosis or systemic inflammation, but low expression in normal mucosa was linked to high serum MMP-8 and IL-8. These findings indicate that tumor necrosis is associated with low TLR4 expression in cancer cells and that low TLR4 expression correlates with a strong systemic inflammatory response. The low TLR2 expression in normal mucosa and its association with systemic inflammation suggest that the normal mucosa may reflect or contribute to the systemic inflammatory response.

Neoadjuvant Chemotherapy Is Associated with Altered Immune Cell Infiltration and an Anti-Tumorigenic Microenvironment in Resected Pancreatic Cancer.

PURPOSE: Neoadjuvant chemotherapy is increasingly administered to patients with resectable or borderline resectable pancreatic ductal adenocarcinoma (PDAC), yet its impact on the tumor immune microenvironment is incompletely understood. DESIGN: We employed quantitative, spatially resolved multiplex immunofluorescence and digital image analysis to identify T-cell subpopulations, macrophage polarization states, and myeloid cell subpopulations in a multi-institution cohort of up-front resected primary tumors (n = 299) and in a comparative set of resected tumors after FOLFIRINOX-based neoadjuvant therapy (n = 36) or up-front surgery (n = 30). Multivariable-adjusted Cox proportional hazards models were used to evaluate associations between the immune microenvironment and patient outcomes. RESULTS: In the multi-institutional resection cohort, immune cells exhibited substantial heterogeneity across patient tumors and were located predominantly in stromal regions. Unsupervised clustering using immune cell densities identified four main patterns of immune cell infiltration. One pattern, seen in 20% of tumors and characterized by abundant T cells (T cell-rich) and a paucity of immunosuppressive granulocytes and macrophages, was associated with improved patient survival. Neoadjuvant chemotherapy was associated with a higher CD8:CD4 ratio, greater M1:M2-polarized macrophage ratio, and reduced CD15+ARG1+ immunosuppressive granulocyte density. Within neoadjuvant-treated tumors, 72% showed a T cell-rich pattern with low immunosuppressive granulocytes and macrophages. M1-polarized macrophages were located closer to tumor cells after neoadjuvant chemotherapy, and colocalization of M1-polarized macrophages and tumor cells was associated with greater tumor pathologic response and improved patient survival. CONCLUSIONS: Neoadjuvant chemotherapy with FOLFIRINOX shifts the PDAC immune microenvironment toward an anti-tumorigenic state associated with improved patient survival.

Prognostic significance of spatial and density analysis of T lymphocytes in colorectal cancer.

BACKGROUND: Although high T cell density is a strong favourable prognostic factor in colorectal cancer, the significance of the spatial distribution of T cells is incompletely understood. We aimed to evaluate the prognostic significance of tumour cell-T cell co-localisation and T cell densities. METHODS: We analysed CD3 and CD8 immunohistochemistry in a study cohort of 983 colorectal cancer patients and a validation cohort (N = 246). Individual immune and tumour cells were identified to calculate T cell densities (to derive T cell density score) and G-cross function values, estimating the likelihood of tumour cells being co-located with T cells within 20 µm radius (to derive T cell proximity score). RESULTS: High T cell proximity score associated with longer cancer-specific survival in both the study cohort [adjusted HR for high (vs. low) 0.33, 95% CI 0.20-0.52, Ptrend < 0.0001] and the validation cohort [adjusted HR for high (vs. low) 0.15, 95% CI 0.05-0.45, Ptrend < 0.0001] and its prognostic value was independent of T cell density score. CONCLUSIONS: The spatial point pattern analysis of tumour cell-T cell co-localisation could provide detailed information on colorectal cancer prognosis, supporting the value of spatial measurement of T cell infiltrates as a novel, robust tumour-immune biomarker.

Immune cell profiles in the tumor microenvironment of early-onset, intermediate-onset, and later-onset colorectal cancer.

BACKGROUND: Despite heightened interest in early-onset colorectal cancer (CRC) diagnosed before age 50, little is known on immune cell profiles of early-onset CRC. It also remains to be studied whether CRCs diagnosed at or shortly after age 50 are similar to early-onset CRC. We therefore hypothesized that immune cell infiltrates in CRC tissue might show differential heterogeneity patterns between three age groups (< 50 "early onset," 50-54 "intermediate onset," ≥ 55 "later onset"). METHODS: We examined 1,518 incident CRC cases with available tissue data, including 35 early-onset and 73 intermediate-onset cases. To identify immune cells in tumor intraepithelial and stromal areas, we developed three multiplexed immunofluorescence assays combined with digital image analyses and machine learning algorithms, with the following markers: (1) CD3, CD4, CD8, CD45RO (PTPRC), and FOXP3 for T cells; (2) CD68, CD86, IRF5, MAF, and MRC1 (CD206) for macrophages; and (3) ARG1, CD14, CD15, CD33, and HLA-DR for myeloid cells. RESULTS: Although no comparisons between age groups showed statistically significant differences at the stringent two-sided α level of 0.005, compared to later-onset CRC, early-onset CRC tended to show lower levels of tumor-infiltrating lymphocytes (P = 0.013), intratumoral periglandular reaction (P = 0.025), and peritumoral lymphocytic reaction (P = 0.044). Compared to later-onset CRC, intermediate-onset CRC tended to show lower densities of overall macrophages (P = 0.050), M1-like macrophages (P = 0.062), CD14+HLA-DR+ cells (P = 0.015), and CD3+CD4+FOXP3+ cells (P = 0.039). CONCLUSIONS: This hypothesis-generating study suggests possible differences in histopathologic lymphocytic reaction patterns, macrophages, and regulatory T cells in the tumor microenvironment by age at diagnosis.

Smoking and Incidence of Colorectal Cancer Subclassified by Tumor-Associated Macrophage Infiltrates.

BACKGROUND: Biological evidence indicates that smoking can influence macrophage functions and polarization, thereby promoting tumor evolution. We hypothesized that the association of smoking with colorectal cancer incidence might differ by macrophage infiltrates. METHODS: Using the Nurses' Health Study and the Health Professionals Follow-up Study, we examined the association of smoking with incidence of colorectal cancer subclassified by macrophage counts. Multiplexed immunofluorescence (for CD68, CD86, IRF5, MAF, and MRC1 [CD206]) combined with digital image analysis and machine learning was used to identify overall, M1-polarized, and M2-polarized macrophages in tumor. We used inverse-probability-weighted multivariable Cox proportional hazards regression models to control for potential confounders and selection bias because of tissue data availability. All statistical tests were 2-sided. RESULTS: During follow-up of 131 144 participants (3 648 370 person-years), we documented 3092 incident colorectal cancer cases, including 871 cases with available macrophage data. The association of pack-years smoked with colorectal cancer incidence differed by stromal macrophage densities (Pheterogeneity = .003). Compared with never smoking, multivariable-adjusted hazard ratios (95% confidence interval) for tumors with low macrophage densities were 1.32 (0.97 to 1.79) for 1-19 pack-years, 1.31 (0.92 to 1.85) for 20-39 pack-years, and 1.74 (1.26 to 2.41) for 40 or more pack-years (Ptrend = .004). In contrast, pack-years smoked was not statistically significantly associated with the incidence of tumors having intermediate or high macrophage densities (Ptrend > .009, with an α level of .005). No statistically significant differential association was found for colorectal cancer subclassified by M1-like or M2-like macrophages. CONCLUSIONS: The association of smoking with colorectal cancer incidence is stronger for tumors with lower stromal macrophage counts. Our findings suggest an interplay of smoking and macrophages in colorectal carcinogenesis.

Spatial Organization and Prognostic Significance of NK and NKT-like Cells via Multimarker Analysis of the Colorectal Cancer Microenvironment.

Although tumor-infiltrating T cells hold a beneficial prognostic role in colorectal cancer, other lymphocytic populations are less characterized. We developed a multiplexed immunofluorescence assay coupled with digital image analysis and machine learning to identify natural killer (NK) cells (NCAM1+CD3-), natural killer T-like (NKT-like) cells (NCAM1+CD3+), and T cells (NCAM1-CD3+) within the PTPRC+ (CD45+) cell population and to measure their granzyme B (GZMB; cytotoxicity marker) and FCGR3A (CD16a; NK-cell maturity marker) expression. We evaluated immune cell densities and spatial configuration in 907 incident colorectal carcinoma cases within two prospective cohort studies. We found that T cells were approximately 100 times more abundant than NK and NKT-like cells. Overall, NK cells showed high GZMB expression and were located closer to tumor cells than T and NKT-like cells. In T and NKT-like cells, GZMB expression was enriched in cells in closer proximity to tumor cells. Higher densities of both T and NKT-like cells associated with longer cancer-specific survival, independent of potential confounders (P trend < 0.0007). Higher stromal GZMB+ and FCGR3A+ NK-cell densities associated with longer cancer-specific survival (P trend < 0.003). For T and NKT-like cells, greater proximity to tumor cells associated with longer cancer-specific survival (P trend < 0.0001). These findings indicate that cytotoxic NCAM1+CD3-GZMB+ NK cells and NCAM1+CD3+ NKT-like cells are relatively rare lymphocytic populations within the colorectal cancer microenvironment and show distinct spatial configuration and associations with patient outcome. The results highlight the utility of a quantitative multimarker assay for in situ, single-cell immune biomarker evaluation and underscore the importance of spatial context for tumor microenvironment characterization.

Microenvironment drives cell state, plasticity, and drug response in pancreatic cancer.

Prognostically relevant RNA expression states exist in pancreatic ductal adenocarcinoma (PDAC), but our understanding of their drivers, stability, and relationship to therapeutic response is limited. To examine these attributes systematically, we profiled metastatic biopsies and matched organoid models at single-cell resolution. In vivo, we identify a new intermediate PDAC transcriptional cell state and uncover distinct site- and state-specific tumor microenvironments (TMEs). Benchmarking models against this reference map, we reveal strong culture-specific biases in cancer cell transcriptional state representation driven by altered TME signals. We restore expression state heterogeneity by adding back in vivo-relevant factors and show plasticity in culture models. Further, we prove that non-genetic modulation of cell state can strongly influence drug responses, uncovering state-specific vulnerabilities. This work provides a broadly applicable framework for aligning cell states across in vivo and ex vivo settings, identifying drivers of transcriptional plasticity and manipulating cell state to target associated vulnerabilities.

Neoadjuvant Selicrelumab, an Agonist CD40 Antibody, Induces Changes in the Tumor Microenvironment in Patients with Resectable Pancreatic Cancer.

PURPOSE: CD40 activation is a novel clinical opportunity for cancer immunotherapy. Despite numerous active clinical trials with agonistic CD40 monoclonal antibodies (mAb), biological effects and treatment-related modulation of the tumor microenvironment (TME) remain poorly understood. PATIENTS AND METHODS: Here, we performed a neoadjuvant clinical trial of agonistic CD40 mAb (selicrelumab) administered intravenously with or without chemotherapy to 16 patients with resectable pancreatic ductal adenocarcinoma (PDAC) before surgery followed by adjuvant chemotherapy and CD40 mAb. RESULTS: The toxicity profile was acceptable, and overall survival was 23.4 months (95% confidence interval, 18.0-28.8 months). Based on a novel multiplexed immunohistochemistry platform, we report evidence that neoadjuvant selicrelumab leads to major differences in the TME compared with resection specimens from treatment-naïve PDAC patients or patients given neoadjuvant chemotherapy/chemoradiotherapy only. For selicrelumab-treated tumors, 82% were T-cell enriched, compared with 37% of untreated tumors (P = 0.004) and 23% of chemotherapy/chemoradiation-treated tumors (P = 0.012). T cells in both the TME and circulation were more active and proliferative after selicrelumab. Tumor fibrosis was reduced, M2-like tumor-associated macrophages were fewer, and intratumoral dendritic cells were more mature. Inflammatory cytokines/sec CXCL10 and CCL22 increased systemically after selicrelumab. CONCLUSIONS: This unparalleled examination of CD40 mAb therapeutic mechanisms in patients provides insights for design of subsequent clinical trials targeting CD40 in cancer.

Prognostic significance of myeloid immune cells and their spatial distribution in the colorectal cancer microenvironment.

BACKGROUND: Myeloid cells represent an abundant yet heterogeneous cell population in the colorectal cancer microenvironment, and their roles remain poorly understood. METHODS: We used multiplexed immunofluorescence combined with digital image analysis to identify CD14+ monocytic and CD15+ granulocytic cells and to evaluate their maturity (HLA-DR and CD33), immunosuppressive potential (ARG1) and proximity to cytokeratin (KRT)-positive tumor cells in 913 colorectal carcinomas. Using covariate data of 4465 incident colorectal cancers in two prospective cohort studies, the inverse probability weighting method was used with multivariable-adjusted Cox proportional hazards models to assess cancer-specific mortality according to ordinal quartiles (Q1-Q4) of myeloid cell densities. Immune cell-tumor cell proximity was measured with the nearest neighbor method and the G-cross function, which determines the likelihood of any tumor cell having at least one immune cell of the specified type within a certain radius. RESULTS: Higher intraepithelial (Ptrend=0.0002; HR for Q4 (vs Q1), 0.48, 95% CI 0.31 to 0.76) and stromal (Ptrend <0.0001; HR for Q4 (vs Q1), 0.42, 95% CI 0.29 to 0.63) densities of CD14+HLA-DR+ cells were associated with lower colorectal cancer-specific mortality while, conversely, higher intraepithelial densities of CD14+HLA-DR- cells were associated with higher colorectal cancer-specific mortality (Ptrend=0.0003; HR for Q4 (vs Q1), 1.78, 95% CI 1.25 to 2.55). Spatial analyses indicated that CD15+ cells were located closer to tumor cells than CD14+ cells, and CD14+HLA-DR+ cells were closer to tumor than CD14+HLA-DR- cells (p<0.0001). The G-cross proximity measurement, evaluating the difference in the likelihood of any tumor cell being colocated with at least one CD14+HLA-DR+ cell versus CD14+HLA-DR- cell within a 20 µm radius, was associated with lower colorectal cancer-specific mortality (Ptrend <0.0001; HR for Q4 (vs Q1), 0.37, 95% CI 0.24 to 0.57). CONCLUSIONS: Myeloid cell populations occur in spatially distinct distributions and exhibit divergent, subset-specific prognostic significance in colorectal cancer, with mature CD14+HLA-DR+ and immature CD14+HLA-DR- monocytic phenotypes most notably showing opposite associations. These results highlight the prognostic utility of multimarker evaluation of myeloid cell infiltrates and reveal a previously unrecognized degree of spatial organization for myeloid cells in the immune microenvironment.

Leukocyte Heterogeneity in Pancreatic Ductal Adenocarcinoma: Phenotypic and Spatial Features Associated with Clinical Outcome.

Immunotherapies targeting aspects of T cell functionality are efficacious in many solid tumors, but pancreatic ductal adenocarcinoma (PDAC) remains refractory to these treatments. Deeper understanding of the PDAC immune ecosystem is needed to identify additional therapeutic targets and predictive biomarkers for therapeutic response and resistance monitoring. To address these needs, we quantitatively evaluated leukocyte contexture in 135 human PDACs at single-cell resolution by profiling density and spatial distribution of myeloid and lymphoid cells within histopathologically defined regions of surgical resections from treatment-naive and presurgically (neoadjuvant)-treated patients and biopsy specimens from metastatic PDAC. Resultant data establish an immune atlas of PDAC heterogeneity, identify leukocyte features correlating with clinical outcomes, and, through an in silico study, provide guidance for use of PDAC tissue microarrays to optimally measure intratumoral immune heterogeneity. Atlas data have direct applicability as a reference for evaluating immune responses to investigational neoadjuvant PDAC therapeutics where pretherapy baseline specimens are not available. SIGNIFICANCE: We provide a phenotypic and spatial immune atlas of human PDAC identifying leukocyte composition at steady state and following standard neoadjuvant therapies. These data have broad utility as a resource that can inform on leukocyte responses to emerging therapies where baseline tissues were not acquired.This article is highlighted in the In This Issue feature, p. 1861.

Association of Fusobacterium nucleatum with Specific T-cell Subsets in the Colorectal Carcinoma Microenvironment.

PURPOSE: While evidence indicates that Fusobacterium nucleatum (F. nucleatum) may promote colorectal carcinogenesis through its suppressive effect on T-cell-mediated antitumor immunity, the specific T-cell subsets involved remain uncertain. EXPERIMENTAL DESIGN: We measured F. nucleatum DNA within tumor tissue by quantitative PCR on 933 cases (including 128 F. nucleatum-positive cases) among 4,465 incident colorectal carcinoma cases in two prospective cohorts. Multiplex immunofluorescence combined with digital image analysis and machine learning algorithms for CD3, CD4, CD8, CD45RO (PTPRC isoform), and FOXP3 measured various T-cell subsets. We leveraged data on Bifidobacterium, microsatellite instability (MSI), tumor whole-exome sequencing, and M1/M2-type tumor-associated macrophages [TAM; by CD68, CD86, IRF5, MAF, and MRC1 (CD206) multimarker assay]. Using the 4,465 cancer cases and inverse probability weighting method to control for selection bias due to tissue availability, multivariable-adjusted logistic regression analysis assessed the association between F. nucleatum and T-cell subsets. RESULTS: The amount of F. nucleatum was inversely associated with tumor stromal CD3+ lymphocytes [multivariable OR, 0.47; 95% confidence interval (CI), 0.28-0.79, for F. nucleatum-high vs. -negative category; P trend = 0.0004] and specifically stromal CD3+CD4+CD45RO+ cells (corresponding multivariable OR, 0.52; 95% CI, 0.32-0.85; P trend = 0.003). These relationships did not substantially differ by MSI status, neoantigen load, or exome-wide tumor mutational burden. F. nucleatum was not significantly associated with tumor intraepithelial T cells or with M1 or M2 TAMs. CONCLUSIONS: The amount of tissue F. nucleatum is associated with lower density of stromal memory helper T cells. Our findings provide evidence for the interactive pathogenic roles of microbiota and specific immune cells.

Composition, Spatial Characteristics, and Prognostic Significance of Myeloid Cell Infiltration in Pancreatic Cancer.

PURPOSE: Although abundant myeloid cell populations in the pancreatic ductal adenocarcinoma (PDAC) microenvironment have been postulated to suppress antitumor immunity, the composition of these populations, their spatial locations, and how they relate to patient outcomes are poorly understood. EXPERIMENTAL DESIGN: To generate spatially resolved tumor and immune cell data at single-cell resolution, we developed two quantitative multiplex immunofluorescence assays to interrogate myeloid cells (CD15, CD14, ARG1, CD33, HLA-DR) and macrophages [CD68, CD163, CD86, IFN regulatory factor 5, MRC1 (CD206)] in the PDAC tumor microenvironment. Spatial point pattern analyses were conducted to assess the degree of colocalization between tumor cells and immune cells. Multivariable-adjusted Cox proportional hazards regression was used to assess associations with patient outcomes. RESULTS: In a multi-institutional cohort of 305 primary PDAC resection specimens, myeloid cells were abundant, enriched within stromal regions, highly heterogeneous across tumors, and differed by somatic genotype. High densities of CD15+ARG1+ immunosuppressive granulocytic cells and M2-polarized macrophages were associated with worse patient survival. Moreover, beyond cell density, closer proximity of M2-polarized macrophages to tumor cells was strongly associated with disease-free survival, revealing the clinical significance and biologic importance of immune cell localization within tumor areas. CONCLUSIONS: A diverse set of myeloid cells are present within the PDAC tumor microenvironment and are distributed heterogeneously across patient tumors. Not only the densities but also the spatial locations of myeloid immune cells are associated with patient outcomes, highlighting the potential role of spatially resolved myeloid cell subtypes as quantitative biomarkers for PDAC prognosis and therapy.

The Prognostic Role of Macrophage Polarization in the Colorectal Cancer Microenvironment.

Macrophages are among the most common cells in the colorectal cancer microenvironment, but their prognostic significance is incompletely understood. Using multiplexed immunofluorescence for CD68, CD86, IRF5, MAF, MRC1 (CD206), and KRT (cytokeratins) combined with digital image analysis and machine learning, we assessed the polarization spectrum of tumor-associated macrophages in 931 colorectal carcinomas. We then applied Cox proportional hazards regression to assess prognostic survival associations of intraepithelial and stromal densities of M1-like and M2-like macrophages while controlling for potential confounders, including stage and microsatellite instability status. We found that high tumor stromal density of M2-like macrophages was associated with worse cancer-specific survival, whereas tumor stromal density of M1-like macrophages was not significantly associated with better cancer-specific survival. High M1:M2 density ratio in tumor stroma was associated with better cancer-specific survival. Overall macrophage densities in tumor intraepithelial or stromal regions were not prognostic. These findings suggested that macrophage polarization state, rather than their overall density, was associated with cancer-specific survival, with M1- and M2-like macrophage phenotypes exhibiting distinct prognostic roles. These results highlight the utility of a multimarker strategy to assess the macrophage polarization at single-cell resolution within the tumor microenvironment.