Abstract
Introduction
Colorectal cancer (CRC) is a malignancy characterized by strong associations with chronic inflammation. While tumor-infiltrating immune responses have been extensively studied, systemic alterations in innate immune cells, particularly monocytes and macrophages, remain underexplored. Interleukin-1β (IL-1β), a key pro-inflammatory cytokine, plays a pivotal role in both systemic inflammation and tumor microenvironment modulation. This study aimed to evaluate IL-1β secretion by monocyte-derived macrophages from CRC patients and assess its association with clinicopathological features.
Methods
Peripheral blood monocytes were isolated from 20 CRC patients and differentiated into macrophages in vitro using M-CSF. Macrophages were then stimulated with lipopolysaccharide (LPS) under three conditions: single stimulation, repeated stimulation, and no stimulation. IL-1β concentrations in culture supernatants were measured by ELISA. Patients were stratified by tumor grade and stage, and non-parametric statistical tests (Mann–Whitney U, Kruskal–Wallis) were used to assess differences in cytokine secretion across groups.
Results
Macrophage IL-1β secretion was significantly higher in patients with tumors extending beyond the muscularis propria compared to those with intact muscularis propria (median 30.33 vs 15.39 pg/mL, p = 0.0381). Patients with stage IV CRC exhibited a markedly greater fold increase in IL-1β secretion after LPS stimulation than those with stage I (9.08-fold vs 1.63-fold, p = 0.023). Although differences by tumor grade did not reach statistical significance, a trend toward increased IL-1β secretion was observed in higher-grade tumors.
Conclusion
Systemic IL-1β secretion by monocyte-derived macrophages is enhanced in CRC patients with advanced disease, suggesting tumor-driven reprogramming of innate immune responses. These findings support IL-1β as a potential biomarker of tumor progression and a candidate target for immunomodulatory strategies in CRC. Given the modest sample size (n = 20) and the ex vivo design, our conclusions are preliminary and hypothesis-generating. Validation in an independent, larger cohort is warranted.
Introduction
Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality worldwide. Moreover, CRC is a malignancy whose pathogenesis is closely associated with inflammation. It is well established that the development of certain CRCs is linked to pre-existing chronic inflammation of the colon. 1 Inflammation is not merely a causative factor, it plays a critical role in cancer progression by promoting tumor proliferation, stromal remodeling, angiogenesis, invasion, and immune evasion.1,2
A significant but insufficiently studied aspect of CRC pathogenesis is its systemic impact on the production, differentiation, and function of innate immune cells. This influence occurs through multiple mechanisms, including cell-to-cell communication via molecular signals and metabolic alterations. While numerous studies have established that blood levels of soluble inflammatory markers, such as C-reactive protein, interleukin-6, and soluble tumor necrosis factor receptor 2 are associated with disease prognosis,3,4 there remains a notable lack of research focused on changes in innate immune cells outside the tumor microenvironment. Nonetheless, emerging data indicate a profound tumor-driven influence on systemic immune processes, including the emergence of novel cell populations within the bone marrow hematopoietic system such as myeloid-derived suppressor cells (MDSCs), 5 as well as transcriptomic reprogramming and phenotypic shifts in circulating monocytes.6–8
Among mediators of innate immunity, interleukin-1β (IL-1β) occupies a central position. Its production in monocytes/macrophages is governed by activation of inflammasomes—primarily NLRP3—and caspase-1, culminating in the initiation of pro-inflammatory signaling cascades.9,10 The influence of this cytokine extends both within the tumor microenvironment and systemically. 11 The functions of inflammasomes in CRC are context-dependent and may either promote tumor growth or exert protective effects, underscoring the need to evaluate inflammation at distinct biological levels. IL-1 signaling contributes to the maintenance of chronic inflammation, angiogenesis, and immunosuppression; it can upregulate PD-L1 expression and recruit myeloid cells with immunosuppressive phenotypes. 12 Within the tumor stroma—including IL1R1-positive fibroblasts—this axis actively modulates tumor growth and invasion. 13
Most clinical studies evaluate circulating cytokine levels in blood, however, in our view, analyses centered on cellular immune populations are more informative. This is particularly pertinent for IL-1β, as monocytes and macrophages constitute its principal sources.14,15
Based on these considerations, we hypothesized that peripheral blood monocytes from patients with advanced-stage CRC exhibit altered reactivity. Specifically, we posited that monocyte-derived macrophages from such patients display an augmented IL-1β response to primary or secondary (re)stimulation with lipopolysaccharide (LPS), relative to early-stage disease, and that these differences may be associated with key clinico-morphological characteristics. The objective of the study was to quantitatively assess basal and inducible IL-1β secretion under these conditions and to determine its relationship with depth of invasion, pathological stage, and tumor differentiation grade.
Understanding the systemic effects of CRC on innate immune responses holds considerable practical importance. Innate immune mechanisms may provide new opportunities for identifying therapeutic targets for immunomodulation or for developing non-invasive biomarkers to monitor tumor progression offering an alternative to conventional approaches that require surgical access to tumor tissue. 16 In this context, investigating the tumor's systemic impact on innate immune cells, is both actual for cell-biology investigation and clinically relevant.
We employed a standardized ex vivo monocyte-derived macrophage model to assess the functional IL-1β secretion response under controlled stimulation. This approach complements, but does not replace analysis of tumor-derived macrophages and monocyte-derived myeloid cells in tumor tissue and allows us to isolate the donor-specific component of innate responsiveness while minimizing confounding from cellular admixture.
Methods
Study Participants
The study included 20 individuals with histologically confirmed CRC, classified C18–C20 according to the 10th revision of the International Classification of Diseases (ICD-10). The pathomorphological analysis included determination of tumor histotype, histological tumor grade (G1-G3) and staging according to the TNM classification system (eighth edition, 2017) (Table 1).
CRC Participants Characteristics.
There were the exclusion criteria: age younger than 40 or older than 79 years; prior anti-tumor treatment (including chemotherapy, radiotherapy, or immunotherapy); a history of other malignant tumors within the past five years; presence of concomitant autoimmune or inflammatory diseases; history of hepatitis B, hepatitis C, or HIV infection; psychiatric disorders; alcohol or substance abuse; decompensated renal or hepatic failure; and chronic heart failure classified as NYHA class III–IV. No contemporaneous control arm (healthy or benign) was included by design; analyses were restricted to within-CRC contrasts.
Peripheral Blood Monocyte Culture
To obtain the monocyte fraction from peripheral blood, 30 mL of venous blood was subjected to gradient centrifugation using Ficoll to isolate peripheral blood mononuclear cells (PBMCs). The resulting cell fraction was incubated with CD14 + magnetic microbeads (CD14+ MicroBeads, Miltenyi Biotec Inc., USA) and separated using LS columns (Miltenyi Biotec Inc., USA) according to the manufacturer's protocol.
The ex vivo monocyte-derived macrophage model was chosen to measure a functional cell response as IL-1β secretion under strictly standardized differentiation and stimulation. This design reduces variability from tumor tissue heterogeneity and targets the protein-level effector output, which critically depends on post-transcriptional mechanisms.
For macrophage differentiation conditions, isolated CD14 + monocytes were seeded in triplicate into wells of a multiwell plate 1,000,000 cells/mL in RPMI-1640 medium (Paneco, Russia) supplemented with 10% fetal bovine serum (FBS, Biolot, Russia), 2 mM L-glutamine (Paneco, Russia), penicillin-streptomycin (Paneco, Russia), and macrophage colony-stimulating factor (M-CSF) at a final concentration of 50 ng/mL. Cells were cultured at 37 °C in a humidified atmosphere with 5% CO₂. On day 3 of cultivation, half of the culture medium was replaced with fresh medium of identical composition.
On day 6, the entire medium was aspirated, wells were washed with isotonic phosphate-buffered saline (PBS, pH 7.4), and fresh medium without M-CSF was added. For each peripheral blood monocyte sample, cells were cultured in three separate wells: one without lipopolysaccharide (Lipopolysaccharides from Escherichia coli 0111:B4 (LPS) (Sigma-Aldrich, the USA)) and two with LPS added at a final concentration of 100 ng/mL.
After 24 h (day 7), the culture medium was collected from each well to determine the concentration of IL-1β using enzyme-linked immunosorbent assay (ELISA). The first well (without LPS) represented basal cytokine secretion, while the medium from the second and third wells reflected cytokine levels following the initial LPS stimulation.
Following collection, the wells were washed with isotonic phosphate-buffered saline. The first and second wells were then refilled with fresh medium without LPS, while the third well was refilled with medium containing LPS at a final concentration of 100 ng/mL.
After an additional 24 h (day 8), the culture medium was collected to assess IL-1β concentration by ELISA. Thus, the supernatant from the second well was used to determine IL-1β levels 24 h after the initial LPS stimulation, while the third well was used to evaluate IL-1β secretion following repeated (chronical) LPS stimulation.
ELISA Measurement of IL-1β
The concentration of IL-1β in the culture supernatants was measured using an enzyme-linked immunosorbent assay (ELISA) with a commercial Human IL-1β DuoSet ELISA kit (R&D Systems Inc., USA).
Following six days of cultivation under macrophage-differentiating conditions and complete replacement of the culture medium, the following experimental conditions were assessed:
Basal cytokine level after 24 h of incubation without LPS stimulation (no stimulation);
Cytokine level measured 24 h after the initial addition of LPS (first stimulation);
Basal cytokine level measured 48 h after the first LPS stimulation in cultures that were not restimulated (no restimulation);
Cytokine level after a second 24-h LPS stimulation (restimulation).
Statistical Analysis
This study is a hypothesis-generating ex vivo analysis of innate immune responses. As the biologically grounded primary contrast, we compared patients with tumors confined to the intestinal muscular layer versus those with invasion beyond it; comparisons among individual stages were considered as exploratory.
Statistical analysis was performed using GraphPad Prism version 8.0.2 (GraphPad Software Inc., USA). The distribution of the data was assessed using the Shapiro–Wilk test. We used non-parametric methods: descriptive statistics are presented as median and interquartile range (IQR). The Mann–Whitney U test and Kruskal–Wallis test were used to compare differences between groups.
Ethical Approval and Informed Consent Statements
The study was conducted in accordance with the principles of the 1975 Helsinki Declaration and its 2013 version. The research protocol №5 was approved by the local ethical committee
Results
We compared IL-1β secretion levels by macrophages derived from monocytes of CRC patients under basal conditions, after the first stimulation with LPS, and after the second LPS stimulation. This approach can reflect the functional status of peripheral blood monocytes, which are one of the key contributors to both systemic and local inflammation in cancer patients.
The aim of the study was to investigate the secretion of one of the most valuable pro-inflammatory cytokines IL-1β by monocytes differentiated into their most functionally specialized form – macrophages We investigated it in relation to the clinicopathological features of colorectal cancer individuals.
For this purpose, we assessed IL-1β secretion under basal conditions, following the first LPS stimulation (modeling primary activation of pattern recognition receptor pathways), following the second LPS stimulation (modeling chronic activation of pattern recognition receptor pathways), and between the first and second stimulations, reflecting the degree of inflammatory response attenuation.
Comparison of IL-1β Secretion Across Tumor Grades
We compared IL-1β secretion among patients stratified by tumor grade to analyze differences in absolute levels and relative (fold-change) expression. Pairwise group comparisons were performed using the Mann–Whitney U test. According to our data, there were no statistically significant differences in IL-1β secretion by macrophages under basal conditions or after LPS stimulation among individuals with different tumor grades. The most consistent trend was observed when higher-grade tumors were pooled (G2–G3); however, even in this case the differences did not reach statistical significance (Table 2). Similarly, no significant differences were observed in the relative change of IL-1β secretion between the different culture conditions, even when the higher-grade groups were combined and analyzed as a single category (Table 3).
Macrophage IL-1β Level of Different Tumor Grades.
Relative Change of IL-1β Secretion by Macrophages from CRC Patients with Different Tumor Grades.
Comparison of IL-1β Secretion Across Tumor Stages and Progression
We compared IL-1β secretion among patients stratified by disease stage to assess differences in both absolute levels and relative (fold-change) expression of the cytokine. Statistical significance across stages was evaluated using the Kruskal–Wallis test, followed by pairwise comparisons with the Mann–Whitney U test.
Comparison of IL-1β secretion levels by macrophages from individuals stratified by disease stages also revealed no statistically significant differences (Table 4).
Macrophage IL-1β Level of Different CRC Stages.
However, comparison of cytokine secretion by macrophages from patients with tumors limited to the muscularis propria of the colon (stage I) and those with tumor invasion beyond the muscularis propria (stages II-IV) revealed a statistically significant increase in IL-1β levels. In patients with CRC extending beyond the muscularis propria, macrophages demonstrated elevated IL-1β secretion upon first LPS stimulation (30.33 [16.74-44.20] pg/mL vs limited to the muscularis propria 15.39 [8.358-25.52] pg/mL) (Table 5).
Relative Change of IL-1β Secretion in CRC Limited to the Muscularis propria Versus Extending Beyond the Muscularis propria.
The fold change in IL-1β secretion by macrophages also varied depending on the stage of the disease. For example, macrophages from patients with stage IV CRC secreted 9.081 (3.686-9.672) times more IL-1β after the first LPS stimulation compared to basal levels, whereas macrophages from patients with stage I CRC showed only a 1.635 (1.424-4.205) increase (Table 6).
Relative Change of IL-1β Secretion by Macrophages from CRC Patients with Different Tumor Stages.
* p = 0.0364 (Mann–Whitney U test)
Discussion
IL-1β is a key pro-inflammatory cytokine that regulates a wide range of biological processes, including the intensity of inflammation, remodeling of the connective tissue matrix, and modulation of immune cell activity by promoting the production of other pro-inflammatory cytokines such as TNF-α, IL-6, IL-8, and IL-17. Moreover, IL-1β plays a significant role in shaping the tumor microenvironment.8,9
Studies of cytokine secretion in colorectal cancer (CRC) patients have primarily focused on cytokine levels in serum or plasma. However, findings regarding plasma IL-1β levels in CRC patients are contradictory. Some authors have reported lower IL-1β levels in early-stage disease (stages I–II) compared to advanced stages. 17 In contrast, other studies have found significantly reduced IL-1β levels in patients with stage IV CRC relative to earlier stages, 18 highlighting the complexity of IL-1β regulation in CRC and the need to examine its secretion within specific components of the innate immune system.
In this context, our study focused on analyzing changes in IL-1β secretion under basal conditions and in response to single and repeated LPS stimulation in macrophages derived from peripheral blood monocytes. This approach allowed us to model different inflammatory states. Basal secretion may reflect the existing inflammatory background in CRC patients, including inflammaging (chronic low-grade inflammation associated with aging and malignancy). The first LPS stimulation models an acute innate immune response triggered by pathogen-associated molecular patterns, while the second stimulation mimics the effects of chronic activation of pattern-recognition pathways.
CRC is known to exert systemic effects on key cellular effectors of inflammation, including altered monocyte function. These monocytes may later differentiate into macrophages within the tumor microenvironment, contributing to local inflammatory processes. Previous studies have shown that IL-1β secretion by macrophages from CRC patients is elevated compared to healthy donors.19,20 These previously published data (healthy vs CRC) provide external context supporting an elevated IL-1β responsiveness in CRC. Furthermore, tumor-driven reprogramming of blood monocytes has been demonstrated.6,21
Our experimental data support the hypothesis of systemic reprogramming of the monocyte–macrophage compartment in CRC, with alterations detectable at least at the level of circulating blood monocytes (Fig. 1).
Experimental Schema and Outcomes Indicating Heightened IL-1β Responsiveness of the Monocyte–Macrophage Axis in Colorectal Cancer. Peripheral Blood CD14+ Monocytes Were Differentiated into M-CSF–Polarized Macrophages and Assayed for IL-1β Under Basal Conditions, After LPS Stimulation, and upon Restimulation. Across Clinicopathological strata, Macrophages from Advanced Disease Consistently Showed Amplified IL-1β Responses, Supporting Systemic Reprogramming of Circulating Monocytes.
Our findings suggest an association between the degree of systemic functional changes in monocytes and the extent of tumor progression. It is probable that such systemic alterations are translated into local processes following the recruitment and differentiation of monocytes into tumor-infiltrating macrophages. We observed a statistically significant increase in IL-1β secretion after the first LPS stimulation by macrophages from patients with tumors extending beyond the muscularis propria of the colon compared to those with tumors limited to the muscularis propria (15.39 (8.358-25.52) pg/mL versus 30.33 (16.74-44.20) pg/mL, respectively). In addition, macrophages from patients with advanced-stage CRC showed a significantly greater fold increase in IL-1β secretion following first LPS stimulation compared to those from patients with stage I disease (9.081 (3.686-9.672)-fold versus 1.635 (1.424-4.205)-fold, respectively).
These changes may contribute to the pro-tumorigenic effects of IL-1β, including the promotion of angiogenesis, maintenance of chronic inflammation, and recruitment of monocytes, neutrophils, and myeloid-derived suppressor cells, which acquire immunosuppressive phenotypes within the tumor microenvironment.
Although no statistically significant differences were observed, we noted a trend toward increased basal IL-1β secretion in patients with high-grade tumors compared to those with low-grade tumors (6.28 (4.140-21.82) versus 4.00 (3.538-4.440) pg/mL, p = 0.0652).
Limitations: The principal limitation of our investigation was the small sample size (n = 20), which reduces statistical power and external generalizability. While the standardized ex vivo design and unified experimental protocol enhance internal validity, they do not substitute for external validation. Accordingly, the reported associations should be regarded as preliminary and hypothesis-generating, pending confirmation in an independent, larger cohort.
Our findings were obtained in a standardized ex vivo monocyte-derived macrophages (MDM) model and reflect donor-specific readiness of the monocyte lineage to secrete IL-1β upon primary/ restimulation. We acknowledge that tumor-associated macrophage (TAM) phenotypes are microenvironment-educated and not identical to MDM; therefore, external generalizability is limited. We consider our approach complementary to tissue-based studies (eg, scRNA-seq), providing functional causal stimulus–response evidence at the level of secreted cytokines. Additional clinical interpretability can be complemented with direct IL-1β measurements in blood and/or tumor tissue.
Conclusion
Our findings support the hypothesis that the monocyte–macrophage compartment in colorectal cancer undergoes systemic reprogramming, manifesting as heightened IL-1β responsiveness of monocyte-derived macrophages under primary and repeated LPS stimulation. Although several between-group comparisons did not reach statistical significance, consistent response patterns across clinicopathological strata indicate that functional assays of myeloid cells may be more informative than static measurements of circulating cytokines. Future work should validate these observations in larger, prospective cohorts, incorporate single-cell and metabolic profiling to define mechanistic drivers, and test the prognostic and predictive value of myeloid IL-1β responsiveness in the context of disease stage, depth of invasion, and differentiation grade.
Our results are consistent with a reproducible innate response pattern in a standardized ex vivo model. Given the small sample size, these conclusions should be considered preliminary.
Footnotes
Ethical Approval and Informed Consent Statements
The study was conducted in accordance with the principles of the 1975 Helsinki Declaration and its 2013 version. The research protocol №5 was approved by the local ethical committee of the Petrovsky National Research Center of Surgery on May 23, 2025. All study participants provided written informed consent.
Author Contributions
Author Contributions: Nikolay Sadykhov, Nikolay Shakhpazyan, Liudmila Mikhaleva, Arkady Bedzhanyan and Alexander Orekhov—conceptualization, investigation, visualization, writing—original draft preparation, editing, and supervision, Aydin Bumbazhay Alexandra Konyukova Ksenia Maslyonkina and Andrey Kontorschikov—review and editing. All authors have read and agreed to the published version of the manuscript.
Funding
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Russian Science Foundation, (grant number 23-15-00140).
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Data Availability Statement
All data and materials are available upon reasonable request. Ad-dress the request to N.K.S. (Nikolay K. Shakhpazyan) (email: nshakhpazyan@gmail.com) Avtsyn Research Institute of Human Morphology, Petrovsky National Research Center of Surgery, 119435 Moscow, Russia.