Colorectal cancer (CRC) ranks third in cancer incidence and second in cancer deaths worldwide, accounting for an estimated 2.2 million new cases and 1 million deaths each year. Despite improvements in treatment, many patients still face poor outcomes, making the search for new biological targets critically important.
A central challenge in treating CRC is the tumor immune microenvironment (TME) -- a complex network of immune cells, proteins, and signals surrounding the tumor. In many CRC cases, this environment is immunosuppressive, meaning it actually protects the tumor from the body's natural defenses rather than helping to destroy it.
CALB2 (Calbindin 2) is a calcium-binding protein encoded by the CALB2 gene. While it plays an important role in the nervous system as a calcium buffer and signal sensor, its function in colorectal cancer had not been well understood. Prior research linked CALB2 to lung, ovarian, and pancreatic cancers, prompting researchers to investigate its role in CRC.
This study set out to determine whether CALB2 acts as a driver of CRC progression, whether it shapes the tumor's immune environment, and whether it could serve as a new biomarker or therapeutic target for patients with this disease.
Researchers used large public databases -- including TCGA (The Cancer Genome Atlas) and GEO (Gene Expression Omnibus) -- to analyze CALB2 gene expression across hundreds of CRC patient samples and examine how it relates to patient survival and disease stage.
In the laboratory, the team worked with multiple CRC cell lines (HCT116, HT-29, SW480, SW620, DLD-1, and HCT8) and used small interfering RNA (siRNA) to knock down CALB2 expression, and overexpression plasmids to increase it. This allowed them to observe what happened to cancer cell behavior when CALB2 was turned up or down.
Cell experiments included tests for proliferation (how fast cells grow), migration (how cells spread), colony formation (how cells establish new clusters), and apoptosis (programmed cell death). Techniques such as CCK8, EdU staining, transwell assays, and flow cytometry were used to measure each of these behaviors.
To examine effects on the immune environment, the researchers conducted co-culture experiments -- growing tumor cells alongside immune cells (T cells and macrophages) -- and used multiplex immunohistochemistry (mIHC) on actual patient tissue samples to visualize immune cell patterns in the tumor microenvironment.
Statistical analyses included Kaplan-Meier survival curves, Cox regression models to identify independent prognostic factors, and pathway enrichment analyses (GSEA-KEGG and GSEA-GO) to identify the molecular pathways most associated with CALB2 activity.
Analysis across multiple patient datasets consistently showed that CALB2 is highly expressed in colorectal tumor tissue compared with normal adjacent tissue. Its expression was significantly higher in patients with more advanced disease stages -- including deeper tumor invasion (T3/T4), spread to lymph nodes (N1/N2/N3), and distant metastasis (M1).
Patients with high CALB2 expression had significantly shorter overall survival, disease-free survival, and progression-free survival compared to those with low expression. Multivariate Cox regression confirmed that CALB2 expression was an independent predictor of poor outcome -- meaning it predicted worse prognosis even after accounting for other clinical factors like tumor stage and age.
In the researchers' own cohort of 36 CRC patients, CALB2 expression was positively correlated with invasion depth, lymph node involvement, and distant metastasis (all statistically significant). Immunofluorescence imaging directly confirmed higher CALB2 protein levels in tumor tissue versus normal tissue in these patients.
CALB2 was also notably elevated in patients carrying a BRAF mutation, a known marker of aggressive CRC, suggesting possible connections between CALB2 activity and specific molecular subtypes of the disease.
Laboratory experiments confirmed that CALB2 protein was elevated in multiple CRC cell lines (HT-29, SW480, SW620, HCT116) compared with normal colon cells. When CALB2 was silenced using siRNA, cancer cells showed significantly reduced viability, fewer colonies, and decreased proliferation in both CCK8 and EdU assays.
Conversely, overexpressing CALB2 had the opposite effect: cancer cells grew faster, formed more colonies, and divided more actively. Flow cytometry revealed that silencing CALB2 increased the fraction of cells undergoing apoptosis (programmed cell death), while overexpression of CALB2 reduced apoptosis -- confirming its role in keeping cancer cells alive.
In migration assays, overexpressing CALB2 significantly enhanced cancer cells' ability to move and invade, a key early step in metastasis. Western blot analysis showed that CALB2 promoted this migratory behavior by increasing the expression of epithelial-mesenchymal transition (EMT) proteins -- N-cadherin, Slug, and Snail1 -- that help cancer cells detach from the primary tumor and travel to new sites.
Taken together, these results position CALB2 as an oncogene in CRC: a gene whose elevated activity actively promotes cancer cell survival, division, and the capacity to spread.
Bioinformatics analysis of patient datasets using multiple immune infiltration algorithms (CIBERSORT, TIMER, xCell, EPIC, Quantiseq) showed that high CALB2 expression strongly correlated with increased M2 macrophage and fibroblast infiltration, and decreased infiltration of CD8+ T cells -- the immune cells responsible for killing cancer cells directly.
This pattern was confirmed directly in patient tissue samples using multiplex immunohistochemistry (mIHC). Tumor tissues from high-CALB2 patients showed more M2 macrophages (CD11b+CD163+ cells) and more cancer-associated fibroblasts (CAFs, marked by FAP and alpha-SMA), while having significantly fewer CD8+ T cells in the tumor interior.
M2 macrophages are a type of immune cell that, when recruited to tumors, suppress the anti-cancer immune response rather than support it. Cancer-associated fibroblasts build physical barriers in the tumor that prevent immune cells from penetrating. Together, these two cell types create what researchers call a cold tumor immune microenvironment -- a state where the immune system is effectively locked out.
Chemokine pathway analysis revealed that CALB2 was strongly associated with several immunosuppressive chemokines, including CCL18, CCL13, CCL8, and CCL5 -- chemical signals used by tumors to recruit and manipulate immune cells to their advantage.
In co-culture experiments, tumor cells with CALB2 knocked down were placed together with activated immune cells (PBMCs, which include T cells). The results showed that CD8+ T cells produced significantly more anti-tumor signals -- including TNF-alpha, Granzyme B (GzmB), and IFN-gamma -- when CALB2 was silenced in the cancer cells, demonstrating restored T cell activity.
Killing assays confirmed that silencing CALB2 significantly enhanced the ability of T cells to destroy tumor cells, as measured by both cell viability (CCK8 assay) and LDH release (a marker of cell death). This enhancement was most dramatic at higher immune-to-tumor cell ratios.
When macrophages were cultured with conditioned medium (fluid containing secreted factors) from CALB2-overexpressing tumor cells, they showed increased expression of M2 macrophage markers (CD163, CD206, CD200R, VEGF, IL-4) and decreased expression of M1 markers (CD86, IL-6, IFN-gamma, TNF-alpha). M1 macrophages normally fight tumors, while M2 macrophages support tumor growth -- CALB2 essentially flipped this switch toward the pro-tumor M2 state.
Similar experiments showed that CALB2 overexpression in tumor cells also activated cancer-associated fibroblasts (CAFs), as measured by increased expression of ACTA2 (encoding alpha-SMA) and FAP -- two key markers of fibroblast activation.
To understand the molecular mechanism behind CALB2's immunosuppressive effects, the team focused on the STAT3 signaling pathway -- a key regulator of both macrophage polarization and fibroblast activation in the tumor microenvironment. Western blot analysis confirmed that overexpressing CALB2 increased phosphorylated STAT3 (p-STAT3), the active form of this signaling molecule, while silencing CALB2 decreased p-STAT3 levels.
When a STAT3 inhibitor (Stattic) was added to CALB2-overexpressing cells, the immunosuppressive effects were reversed: fibroblast activation markers (ACTA2 and FAP) were reduced, and M2 macrophage markers (CD206, CD163, CD200R) also declined. This confirmed that CALB2's effects on the immune microenvironment depend on the STAT3 pathway.
Among several chemokines tested, CCL5 showed the most significant increase when CALB2 was overexpressed, and the most significant decrease when CALB2 was silenced. CCL5 is a well-established promoter of M2 macrophage polarization and fibroblast activation. Adding the STAT3 inhibitor reduced CCL5 levels, confirming that CALB2 drives CCL5 secretion through STAT3 activation.
This establishes a clear mechanistic chain: CALB2 activates STAT3, which increases CCL5 secretion, which in turn recruits and polarizes M2 macrophages and activates fibroblasts, collectively building and maintaining the immunosuppressive tumor microenvironment that allows CRC to evade immune attack.
This study provides strong evidence that CALB2 acts as an oncogene in colorectal cancer, with dual roles: directly promoting tumor cell survival, proliferation, and migration, while also engineering an immunosuppressive environment that shields the tumor from immune attack.
Because high CALB2 expression predicts poor prognosis independently of other clinical factors, it has potential as a prognostic biomarker -- a measurable signal that could help doctors identify patients at higher risk and tailor their treatment plans accordingly.
The identification of the CALB2-STAT3-CCL5 axis opens new possibilities for targeted therapy. Drugs that block CALB2 activity, inhibit STAT3, or disrupt CCL5 signaling could potentially reverse the immunosuppressive microenvironment, making CRC tumors more susceptible to immunotherapy approaches such as checkpoint inhibitors and T cell-based treatments.
The authors acknowledge that future studies are needed to validate these findings in larger patient cohorts and in animal models, and to explore whether CALB2-targeting strategies can be safely and effectively combined with existing CRC treatments.