Natural killer cell dysfunction is associated with colorectal cancer with severe COVID-19

Abstract

BACKGROUND

Severe acute respiratory syndrome coronavirus 2 induced coronavirus disease 2019 (COVID-19) has posed a great challenge to public health worldwide and also increased susceptibility to colorectal cancer (CRC). Natural killer (NK) cells serve as the first line of defense in the host’s innate immune system, performing natural killing functions and mediating cytotoxicity against tumors and viruses. Therefore, a better understanding of NK cell cytotoxicity may facilitate the development of treatment strategies for CRC-associated with COVID-19.

AIM

To investigate the cytotoxic killing function of peripheral NK cells in patients with CRC and severe COVID-19 (CRC⁺ patients).

METHODS

The percentages of circulating NK and NKT cells in CRC⁺ and age-matched patients with CRC were analyzed using flow cytometry. NK cell cytotoxic activity (NKCA) and corresponding NK cytotoxic factor (NKCF) activity in peripheral blood mononuclear cells were evaluated using a Real-Time Cell Analyzer.

RESULTS

The numbers and percentage of peripheral NK and NKT cells in patients with CRC⁺ were lower than those in patients with CRC. Additionally, compared to patients with CRC, those with CRC⁺ had lower levels of NKCA and NKCF activity in lysed K562 cells. Positive correlations were observed between NKCA and NK cell numbers, NKCA and NK cell percentages, NKCF activity, and NK cell percentages in patients with CRC⁺. Furthermore, a negative correlation was observed between NKCA and the severity of COVID-19 in patients with CRC. The area under the receiver operating characteristic curve for NKCA was greater than those for the other indices.

CONCLUSION

CRC⁺ is associated with lower levels of peripheral NK cells and impaired natural cytotoxicity, contributing to the immunopathogenesis of severe COVID-19 rather than immune control.

Key Words: Colorectal cancer; COVID-19; Natural killer cells; Natural cytotoxicity; Natural killer cytotoxic factor

Core Tip: Severe acute respiratory syndrome coronavirus 2-induced coronavirus disease 2019 (COVID-19) has posed a great challenge to public health worldwide and has also increased susceptibility to colorectal cancer (CRC). As the first line of defense in the host’s innate immune system, natural killer (NK) cells play a key role in rapidly identifying and eliminating cancerous cells, viruses, and infected cells. In this investigation, patients with CRC and severe COVID-19 had lower levels of peripheral NK cells and impaired natural cytotoxicity, either through direct NK cell cytotoxic activity or NK cytotoxic factor release, thereby contributing to the immunopathogenesis of severe COVID-19 rather than immune control, at least to some extent.

INTRODUCTION

The coronavirus disease 2019 (COVID-19) pandemic was caused by the highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is commonly associated with progressive innate and adaptive immune cell dysfunction and depletion[1,2]. As the first line of defense in the host’s innate immune system, natural killer (NK) cells play a crucial role in rapid identification and immediate elimination of cancerous cells, viruses, and infected cells through direct NK cell-mediated cytotoxicity and the release of NK cytotoxic factor (NKCF), which enhances broader immune responses[3,4]. Disrupting the balance between innate and adaptive immunity, as well as dysfunction, weakens NK cell-mediated cytotoxicity against SARS-CoV-2, contributing to COVID-19 progression[5,6].

Colorectal cancer (CRC) is the third most prevalent malignancy and the second leading cause of cancer-related mortality globally, with almost 1.92 million (9.6%) new cases and approximately 0.9 million (9.3%) deaths in 2022, posing a considerable burden on human well-being[7-9]. According to cancer burden data issued by the National Cancer Centre of China in 2022, CRC was the second most prevalent cancer, accounting for 517100 cases (1.07 per 10 cases) of all newly diagnosed cancers, and the fourth leading cause of mortality, accounting for 240000 cases (0.9 per 10 cases). This burden continues to rise[10]. Patients with CRC are dual-susceptible to SARS-CoV-2 infection, given their immunodeficiency and high expression levels of angiotensin-converting enzyme 2 and transmembrane protease serine 2 in CRC tissues[11,12]. These factors pose an important challenge in the management and treatment of CRC after China eased COVID-19 restrictions[12]. During SARS-CoV-2 infection, the low number of peripheral blood NK cells and reduced NK cell-mediated cytotoxicity have been reported in patients with severe COVID-19, contributing to immune system dysregulation and immunopathogenicity[13,14]. However, data on COVID-19 in patients with CRC remain scarce. Owing to the role of NK cells in antiviral immune response, this study aimed to investigate peripheral NK cells, focusing on their numbers and proportions, NK cell cytotoxic activity (NKCA), and the corresponding NKCF activity released in patients with CRC and severe COVID-19 (CRC⁺ patients).

MATERIALS AND METHODS

Study population

Thirty patients newly diagnosed with CRC were admitted to Tianjin Medical University Cancer Hospital between November 2022 and February 2023. Of the 30 patients with stage I-II CRC, 15 sex- and age-matched patients had their first CRC diagnosis coincide with severe COVID-19 infection. COVID-19 infection was confirmed by the positive detection of SARS-CoV-2 RNA, and its severity was determined based on the Diagnosis and Treatment of New Coronavirus Pneumonia (version 9) issued by the National Health Commission of China[15]. At the time of admission, severe COVID-19 was defined by the following criteria: (1) Respiratory distress with a respiratory rate > 30 breaths/min; (2) Oxygen saturation ≤ 93% in the resting state; (3) The ratio of partial pressure of arterial oxygen (PaO₂) to the fraction of inspired oxygen (FiO₂) ≤ 300 mmHg; and (4) Progressive worsening of clinical symptoms, with chest imaging showing a significant increase in pulmonary lesions (> 50%) within 24 to 48 hours. The inclusion criteria of this study were as follows: Patients aged > 18 years; those with a histopathologically confirmed diagnosis of CRC; and those who had not previously received standard treatments. In addition, patients with conditions that could potentially affect NK cell function, including recent bacterial infection, known hepatitis B or C infections, or a history of autoimmune disorders, were excluded. Patients who consumed tobacco or alcohol were also excluded. The clinical characteristics of the participants are presented in Table 1. All patients provided informed consent, and the study was approved by the Ethics Committee of Tianjin Medical University Cancer Hospital.

Table 1 Characteristic features of study populations.

	CRC patients	CRC+ patients
Number	15	15
Age	61.9 (43-75)	60.0 (43-73)
Gender (male:female)	8:7	8:7
BMI (kg/m2)	25.93 ± 3.48	25.95 ± 3.38
TNM stage
I	10	9
II	5	6
Degree of differentiation
High	1	1
Moderately	14	14

CRC: Colorectal cancer; CRC⁺: Colorectal cancer with severe coronavirus disease 2019; BMI: Body mass index; TNM: Tumor-node-metastasis.

NK cell number and percentage

Peripheral venous blood samples were collected in the morning under fasting conditions. NK cell phenotypes were determined using cell surface markers, such as anti-peridinin chlorophyll protein complex-conjugated anti-CD45, fluorescein isothiocyanate-conjugated anti-CD3, and phycoerythrin-conjugated anti-CD16 and anti CD56. All antibodies were procured from BD Biosciences (San Jose, CA, United States). The leukocyte population was first gated on a Forward Scatter/Side Scatter dot plot, followed by further gating on CD45/Side Scatter. Cells expressing CD3^-CD16/CD56⁺ and CD3⁺CD16/CD56⁺ surface markers were gated as mature NK and NKT cells, respectively. The frequencies of conventional NK and NKT cells were then determined. Flow cytometric analysis was performed using a FACS Calibur flow cytometer with CellQuest software (BD, United States). A representation of the gating strategy for NK and NKT cells is shown in Figure 1. The number of NK and NKT cells was calculated using the total white blood cell and lymphocyte counts obtained from the full blood examination.

Figure 1 Natural killer and natural killer T cells gating strategy from representative histogram of flow cytometric analysis. Graphs represented from left to right: Leukocyte population gating based on Side Scatter and Forward Scatter dot plot; leukocytes gating through Side Scatter and CD45; upper left quadrant represents CD3^-CD16/CD56⁺ natural killer (NK) cell population. upper right quadrant represents CD3⁺CD16/CD56⁺ NKT cell population.

Isolation of peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMCs) were separated from peripheral venous blood using standard density gradient centrifugation with Ficoll-Hypaque (GE Healthcare Bio-Sciences, Uppsala, Sweden) at room temperature. The isolated PBMCs were resuspended in a freezing medium containing 90% fetal bovine serum (Gibco, MA, United States) and 10% dimethyl sulfoxide (Sigma-Aldrich Corp., MO, United States). PBMC aliquots of 5 × 10⁶ cells were cryopreserved in liquid nitrogen until use. One day prior to the experiment, PBMCs were thawed and incubated overnight at 37 °C with 5% CO₂ to deplete adherent monocytes. The non-adherent lymphocytes were then collected and resuspended as effector cells to assess NK cell cytotoxicity in vitro. To assess NKCF activity, 5 × 10⁵ PBMCs were stimulated with 100 μg/mL of phytohaemagglutinin (PHA; Sigma-Aldrich) and cultured at 37 °C with 5% CO₂. Culture supernatants were harvested after 48 hours.

Assessment of NK cells cytotoxic activity

As described in a previous study[16], the NKCA assay was performed using a real-time cell analyzer (RTCA; Roche Applied Science, Germany), which applies an electrical impedance-based approach. First, 6 μg of fibronectin (Sigma-Aldrich) per well was pre-coated onto 16-well chamber slide plate (E-plate) for 1 hour. Thereafter, 2 × 10⁴ K562 cells were seeded into the E-plates at a volume of 100 μL per well, and electrical impedance was measured at 5-minute intervals throughout the culture period until the target cells reached logarithmic growth. Next, PBMCs were inoculated in E plates at a density of 2.5 × 10⁵ cells in a volume of 100 μL per well and cultured at a 12.5:1 ratio of effector-to-target cells (E/T). Electrical impedance was measured at 10-minute intervals throughout the incubation period of 6 hours. The system detects a dimensionless parameter called the cell index (CI). The percentage of cytotoxicity was calculated as follows: NKCA (%) = [1 - CI (cytotoxicity group)/CI (target cell group)] × 100%.

Assessment of NKCF activity

Briefly, 6 μg of fibronectin per well was pre-coated onto a 16-well chamber slide plates (E-plate) for 1 hour. After adding 2 × 10⁴ K562 cells with or without 100 μL PBMC culture supernatants (containing NKCF), the final volume was 200 μL. The E-plates were incubated at 37 °C with 5% CO₂ and monitored using the RTCA system at 15-minute intervals for up to 24 hours. The percentage of cytotoxicity was calculated as follows: NKCF activity (%) = [1 - CI (viability of test)/CI (viability of target cells)] × 100%.

Statistical analysis

Statistical analyses were performed using SPSS software (version 16.0; SPSS Inc., Chicago, IL, United States). Continuous variables were presented as either mean ± SD or median (interquartile range: 25-75). Independent group t-tests were used to compare means of continuous variables with normal distributions, whereas the Mann-Whitney U test was used for continuous variables that were not normally distributed. Correlation analyses were performed using Pearson’s correlation coefficients. In addition, the correlation between NKCA levels and severe COVID-19 was analyzed using Spearman’s correlation test. Receiver operating characteristic (ROC) curves were used to assess the diagnostic efficiency of various indicators. A P value less than 0.05 was considered statistically significant.

RESULTS

Comparison of numbers and percentages of peripheral NK and NKT cells between patients with CRC and those with CRC⁺

In viral challenge settings, NK cells play an indispensable role in early innate immunity and mediate adaptive responses against SARS-CoV-2 infection. The number and percentage of changes in peripheral blood NK and NKT cells are shown in Figure 2. In the comparative analysis of patients with CRC and those with CRC⁺, the numbers and percentages of NK cells in the peripheral blood, which are commonly used as a surface signature for NK cells, were significantly lower in patients with CRC⁺ than in those with CRC (P < 0.05). In addition, the number and percentage of NKT cells, a subpopulation of CD3⁺ T cells co-expressing T-cell antigen receptors and NK cell markers, significantly decreased in patients with CRC⁺.

Figure 2 Comparison of numbers and percentages of peripheral natural killer and natural killer T cells between colorectal cancer and colorectal cancer with severe the coronavirus disease 2019 patients. A: Representative histogram in peripheral blood of colorectal cancer (CRC) and CRC with severe the coronavirus disease 2019 (CRC⁺) patients; B: Comparison of natural killer (NK) and NKT cell numbers between CRC and CRC⁺ patients; C: Comparison of NK and NKT cell percentages between CRC and CRC⁺ patients. ^aP < 0.05 vs CRC⁺via Student's t test; ^bP < 0.05 vs CRC⁺via Mann-Whitney U test; CRC: Colorectal cancer; CRC⁺: Colorectal cancer with severe the coronavirus disease 2019; NK: Natural killer.

Comparison of NKCA and NKCF activity between patients with CRC and those with CRC⁺

To identify peripheral NK cell dysfunction, PBMCs or culture supernatants with K562 cells were co-cultured in vitro for 6 hours and 24 hours, respectively, to investigate NK cell cytotoxicity, including direct NKCA and indirect NKCF activity, using real-time, label-free measurements. The results showed a significant decrease in NKCA from NK cells derived from patients with CRC⁺ (P < 0.01) compared with those from patients with CRC. Notably, a similar decrease was observed in NKCF activity in patients with CRC⁺ compared with that in patients with CRC (P < 0.05), as shown in Figure 3. These results suggest that the impairment of both NKCA and NKCF activity may account for NK cell dysfunction.

Figure 3 Comparison of natural killer cell cytotoxic activity and natural killer cytotoxic factor activity between colorectal cancer and colorectal cancer with severe the coronavirus disease 2019 patients. ^aP < 0.05 vs colorectal cancer with severe the coronavirus disease 2019 (CRC⁺) via Student's t test; ^bP < 0.01 vs CRC⁺via Mann-Whitney U test; CRC: Colorectal cancer; CRC⁺: Colorectal cancer with severe the coronavirus disease 2019; NKCA: Natural killer cell cytotoxic activity; NKCF: Natural killer cytotoxic factor.

Correlation analysis of NK cells and NK cell cytotoxicity in patients with CRC⁺

A significant correlation was observed between peripheral NK cells and cytotoxicity. In patients with CRC⁺ patients, the correlation coefficients between NKCA and NK cell numbers, NKCA and NK cell percentages, NKCF activity, and NK cell numbers were 0.628, 0.619, and 0.525, respectively (Figure 4).

Figure 4 The correlation analysis between natural killer cells and natural killer cell cytotoxicity in colorectal cancer with severe the coronavirus disease 2019 patients. NK: Natural killer; NKCA: Natural killer cell cytotoxic activity; NKCF: Natural killer cytotoxic factor.

Correlation analysis of NKCA and severe COVID-19 in patients with CRC⁺

After performing the Spearman correlation test, a negative correlation was observed between NKCA and severe COVID-19 in patients with CRC (Table 2). In patients with CRC, the correlation coefficient between NKCA and severe COVID-19 was -0.563.

Table 2 Correlation analysis of natural killer cell cytotoxic activity and severe coronavirus disease 2019 in colorectal cancer with severe coronavirus disease 2019 patients.

	CRC patients	CRC+ patients	rs	P value
NKCA (%)	32.02 ± 13.56	17.34 ± 10.07	-0.563	0.001

CRC: Colorectal cancer; CRC⁺: Colorectal cancer with severe coronavirus disease 2019; NKCA: Natural killer cell cytotoxic activity.

ROC curve analysis of NK cells and NK cell cytotoxicity

ROC curves were used to explore the ability of peripheral NK cells and NK cell cytotoxicity to differentiate between patients with CRC and those with CRC⁺ (Table 3, Figure 5). The area under the ROC curve (AUC) for NKCA (0.824, 0.672-0.977) was larger than those for the other indices. The AUC for the NK cell number, NK cell percentage, NKT cell number, NKT cell percentage, and NKCF activity were 0.727, 0.711, 0.720, 0.724, and 0.738, respectively. The optimal cut-off for detecting CRC⁺ in the NKCA test was 16.95%.

Figure 5 Receiver operating characteristic curve analysis of natural killer cells and natural killer cell cytotoxicity between colorectal cancer and colorectal cancer with severe the coronavirus disease 2019 patients. NK: Natural killer; NKCA: Natural killer cell cytotoxic activity; NKCF: Natural killer cytotoxic factor.

Table 3 Area under the curve and 95%CI of the receiver operator characteristic curve.

	AUC	95%CI	P value
NK cells (%)	0.711	0.526-0.896	0.049
NK cells (109/L)	0.727	0.545-0.908	0.034
NKT cells (%)	0.724	0.539-0.910	0.036
NKT cells (109/L)	0.720	0.535-0.905	0.040
NKCA (%)	0.824	0.672-0.977	0.002
NKCF activity (%)	0.738	0.553-0.922	0.026

AUC: Area under the curve; NK: Natural killer; NKCA: Natural killer cell cytotoxic activity; NKCF: Natural killer cytotoxic factor.

DISCUSSION

As broad-spectrum innate immune effectors with natural cytotoxicity, NK cells are known to rapidly eliminate pathogens and malignant cells; however, NK cell-mediated cytotoxic function in the peripheral blood is disrupted during COVID-19 progression, contributing to the immunopathogenesis of severe or critical illness and predicting poor outcomes[17,18]. The present study characterized the parameters of NK cells from patients with CRC and those with CRC⁺ and found a decrease in NK cells in the peripheral blood of patients with CRC⁺, which was accompanied by significant impairment of both direct and indirect NK cell-mediated cytotoxicity.

Circulating NK cells contribute to the immune response against SARS-CoV-2 infection and play an important role in the rapid progression of COVID-19 and its outcomes[19,20]. Our results are consistent with the current literature, where low NK cell numbers and proportions in the peripheral blood are observed in patients with CRC⁺, which is considered unfavorable for eliminating SARS-CoV-2 infected cells and impairing innate immune responses. In addition to NK cells, a similar decrease was observed in NKT cells in patients with CRC⁺ compared to that observed in patients with CRC. This highlights that the reason for this phenomenon was that the circulating NK and NKT populations measured had shifted toward exhaustion in patients with severe COVID-19, which could predispose them to fail to limit viral pathogenesis and confirm the need for monitoring severe COVID-19 patients. Demaria et al[21] further demonstrated that the number and proportion of CD16⁺CD56⁺ mature NK cells in both peripheral blood and lungs were markedly lower in patients with COVID-19 and ARDS, suggesting that the decrease in circulating mature NK cell levels was not a consequence of their migration to the infected lungs. In contrast, a sharp decline in mature and elevated levels of immature NK cells have been reported as the main contributors to lymphopenia in severe/critical patients[22,23].

NK cell-mediated cytotoxic activity is an essential characteristic that reflects the activation state of NK cells, which is achieved not only through direct cell-cell contact dependence but also by releasing NKCF, ultimately killing the targeted cells[24]. In this study, peripheral NKCA from CRC⁺ patients were significantly lower than their levels in CRC patients at 6 hours in vitro, indicating that the attenuated cytotoxic capacity of NK cells directly rendered their dysfunctional states, thereby impeding the ability to scavenge virus-infected cells as a part of the immune escape mechanisms induced by severe COVID-19. The results of the present study are similar to those of previous studies that investigated the immune phenotype of lymphocyte subsets. In those studies, increased expression of programmed cell death 1 (PD-1) on CD8⁺ T cells and NK cells was found to be significantly associated with the severity of COVID-19 infection in patients with cancer[25]. In addition, the expression levels of PD-1 in NK cells signify an exhausted state of NK cells in patients with severe COVID-19[26], which was confirmed by a strong negative correlation between PD-1 and NKCA levels[27]. In addition to this direct NKCA, we observed significantly lower NKCF activity in lysing NK-sensitive K-562 target cells in patients with CRC⁺. To assess NKCF activity, NKCF was induced using the stimulator PHA, as previously described[28]. NKCF can also be induced by tumor targets; however, this appears to be a nonspecific phenomenon because both NK cell-sensitive and NK cell-less sensitive tumor targets stimulate the release of NKCF. Therefore, to standardize the induction of NKCF, PHA was used as the only inducing agents across different samples to ensure consistency, at fixed concentration (100 μg/mL).

This study demonstrated for the first time that depressed NKCF in CRC⁺ cells was related to the impaired release of a soluble cytotoxic factor, NKCF, with the importance of an effective NK cell-mediated cytotoxic reaction, which was at least partly responsible for the suppressed NK cell activity demonstrated in SARS-CoV-2 clearance and immune escape. Furthermore, correlation analysis indicated a significant correlation between the reduction in circulating NK cells and NKCA, including their corresponding NKCF release, indicating that the conversion of highly cytotoxic NK cells into exhausted NK cells may contribute to the progression and severity of COVID-19. Notably, a previous study reported that the decreased frequency of the NK subpopulation under Pool Spike coronavirus (CoV)-2 stimulus in severe CoV suggests an impairment in cytotoxic activity or an attempt to control NK responsiveness against SARS-CoV-2[29]. Overall, the lack of NK cytotoxic activity against target cells may be due to impairment in the secretion and release of cytolytic granule proteins (granzymes and perforin) through NK cell exocytosis[30,31] and the ability to stimulate NKCF release from activated NK cells, which contributes to defective NK cytotoxicity in the cytolytic and/or cytostatic processes against virus elimination in severe cases of COVID-19. Recent evidence from a multicenter retrospective study emphasized that patients with cancer face an uphill challenge with COVID-19, and cancer patients with COVID-19 have more severely dysregulated immune responses than non-cancer patients, accompanied by decreased NK cells and NK cell dysfunction, which may account for the poorer prognosis of cancer patients with COVID-19[32]. Remarkably, the ROC curve for NKCA was larger than those for the other indices, indicating that natural cytotoxic activity was optimal for differentiating CRC⁺ cases from CRC cases. Therefore, monitoring NKCA in patients with CRC⁺ is of great significance for formulating an effective treatment regimen.

Although NK cells were analyzed in patients with CRC and CRC⁺, this study has several limitations. First, the expression of particular subsets of NK cells and their dysregulated receptors, as well as the bioactivity of the secreted cytokines, was not measured. Therefore, further studies are warranted. Second, a small sample size and only early-stage patients with CRC and severe COVID-19 were investigated, and they did not cover the advanced stages of patient assessment, which is required in larger sample sizes to better assess changes in immune disorders. Additionally, to address the limitations of using peripheral blood NK cell data, it is necessary to consider potential differences in tissue-resident NK cell populations and explore the mechanisms underlying the observed changes in immune cell populations and their impact on the microenvironment of cancer cells. For example, a recent study identified novel exhausted T cell CD8⁺ markers in breast cancer[33].

CONCLUSION

Profound differences were observed in peripheral NK and NKT cells in patients with CRC⁺ compared with those in CRC patients, characterized by a decrease in the numbers and percentages of both NK and NKT cells. Additionally, peripheral NK cell dysfunction was demonstrated to significantly impair NK cell-mediated cytotoxicity, either directly through NKCA or the release of NKCF, contributing to the immunopathogenesis of severe COVID-19 rather than immune control. NKCA is an optimal indicator for differentiating between patients with CRC and CRC⁺. Adoptive NK cell immunotherapy or checkpoint inhibitor combinations for patients with CRC⁺ are likely to be important CRC treatment modalities in the foreseeable future[34]. Another important development was the emphasis of some studies on immune cells and cancer biomarkers, which reported many pan-cancer biomarkers that are potentially related to ultrasound characteristics that can be explored in the future for not only this cancer type, but also other cancer types[35-39].

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the students for skillful technical assistance.