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.
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.
Although high T-cell density is a well-established favorable prognostic factor in colorectal cancer, the prognostic significance of tumor-associated plasma cells, neutrophils, and eosinophils is less ...well-defined.
We computationally processed digital images of hematoxylin and eosin (H&E)-stained sections to identify lymphocytes, plasma cells, neutrophils, and eosinophils in tumor intraepithelial and stromal areas of 934 colorectal cancers in two prospective cohort studies. Multivariable Cox proportional hazards regression was used to compute mortality HR according to cell density quartiles. The spatial patterns of immune cell infiltration were studied using the G
function, which estimates the likelihood of any tumor cell in a sample having at least one neighboring immune cell of the specified type within a certain radius. Validation studies were performed on an independent cohort of 570 colorectal cancers.
Immune cell densities measured by the automated classifier demonstrated high correlation with densities both from manual counts and those obtained from an independently trained automated classifier (Spearman's ρ 0.71-0.96). High densities of stromal lymphocytes and eosinophils were associated with better cancer-specific survival
< 0.001; multivariable HR (4th vs 1st quartile of eosinophils), 0.49; 95% confidence interval, 0.34-0.71. High G
area under the curve (AUC
;
= 0.002) and high G
AUC
(
< 0.001) also showed associations with better cancer-specific survival. High stromal eosinophil density was also associated with better cancer-specific survival in the validation cohort (
< 0.001).
These findings highlight the potential for machine learning assessment of H&E-stained sections to provide robust, quantitative tumor-immune biomarkers for precision medicine.
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.
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.
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 (
= 0.004) and 23% of chemotherapy/chemoradiation-treated tumors (
= 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.
This unparalleled examination of CD40 mAb therapeutic mechanisms in patients provides insights for design of subsequent clinical trials targeting CD40 in cancer.
Obesity is a major modifiable risk factor for pancreatic ductal adenocarcinoma (PDAC), yet how and when obesity contributes to PDAC progression is not well understood. Leveraging an autochthonous ...mouse model, we demonstrate a causal and reversible role for obesity in early PDAC progression, showing that obesity markedly enhances tumorigenesis, while genetic or dietary induction of weight loss intercepts cancer development. Molecular analyses of human and murine samples define microenvironmental consequences of obesity that foster tumorigenesis rather than new driver gene mutations, including significant pancreatic islet cell adaptation in obesity-associated tumors. Specifically, we identify aberrant beta cell expression of the peptide hormone cholecystokinin (Cck) in response to obesity and show that islet Cck promotes oncogenic Kras-driven pancreatic ductal tumorigenesis. Our studies argue that PDAC progression is driven by local obesity-associated changes in the tumor microenvironment and implicate endocrine-exocrine signaling beyond insulin in PDAC development.
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•Obesity accelerates oncogenic Kras-driven pancreatic ductal tumorigenesis in mice•Genetic or dietary weight loss intercepts pancreatic cancer progression•Obesity is associated with aberrant pancreatic islet cholecystokinin expression•Islet cholecystokinin overexpression drives pancreatic ductal cancer development
Obesity is an intrinsic driver of PDAC in mice, leading to a remodeling of beta cells to increase CCK secretion and playing a role early in pancreatic cancer development that can be intercepted by weight loss.
Abstract
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.
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
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.
.
Histological lymphocytic reaction is regarded as an independent prognostic marker in colorectal cancer. Considering the lack of adequate statistical power, adjustment for selection bias and ...comprehensive tumour molecular data in most previous studies, we investigated the strengths of the prognostic associations of lymphocytic reaction in colorectal carcinoma by utilising an integrative database of two prospective cohort studies.
We examined Crohn's-like reaction, intratumoural periglandular reaction, peritumoural reaction and tumour-infiltrating lymphocytes in 1465 colorectal carcinoma cases. Using covariate data of 4420 colorectal cancer cases in total, inverse probability-weighted Cox proportional hazard regression model was used to control for selection bias (due to tissue availability) and potential confounders, including stage, MSI status, LINE-1 methylation, PTGS2 and CTNNB1 expression, KRAS, BRAF and PIK3CA mutations, and tumour neoantigen load.
Higher levels of each lymphocytic reaction component were associated with better colorectal cancer-specific survival (P
< 0.002). Compared with cases with negative/low intratumoural periglandular reaction, multivariable-adjusted HRs were 0.55 (95% CI, 0.42-0.71) in cases with intermediate reaction and 0.20 (95% CI, 0.12-0.35) in cases with high reaction. These relationships were consistent in strata of MSI status or neoantigen loads (P
> 0.2).
The four lymphocytic reaction components are prognostic biomarkers in colorectal carcinoma.
While evidence indicates that
(
) may promote colorectal carcinogenesis through its suppressive effect on T-cell-mediated antitumor immunity, the specific T-cell subsets involved remain uncertain.
We ...measured
DNA within tumor tissue by quantitative PCR on 933 cases (including 128
-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
, 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
and T-cell subsets.
The amount of
was inversely associated with tumor stromal CD3
lymphocytes multivariable OR, 0.47; 95% confidence interval (CI), 0.28-0.79, for
-high vs. -negative category;
= 0.0004 and specifically stromal CD3
CD4
CD45RO
cells (corresponding multivariable OR, 0.52; 95% CI, 0.32-0.85;
= 0.003). These relationships did not substantially differ by MSI status, neoantigen load, or exome-wide tumor mutational burden.
was not significantly associated with tumor intraepithelial T cells or with M1 or M2 TAMs.
The amount of tissue
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.
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.
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.
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.
Neoadjuvant chemotherapy with FOLFIRINOX shifts the PDAC immune microenvironment toward an anti-tumorigenic state associated with improved patient survival.
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