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. 2023 Mar 15;46(3):777–791. doi: 10.1007/s13402-023-00794-w

Targeting CD43 optimizes cancer immunotherapy through reinvigorating antitumor immune response in colorectal cancer

Yi-yi Li 1,4, Xin-yu Wang 1,4, Yan Li 1,4, Xiu-mei Wang 1,4, Jing Liao 2, Ying-zhao Wang 3, Hai Hong 4,5, Wei Yi 1,✉, Jun Chen 4,5,6,7,✉
PMCID: PMC12974646  PMID: 36920728

Abstract

Purpose

Colorectal cancer (CRC) is one of the most common malignancies worldwide, with dramatically increasing incidence and mortality for decades. However, current therapeutic strategies for CRC, including chemotherapies and immunotherapies, have only demonstrated limited efficacy. Here, we report a novel immune molecule, CD43, that can regulate the tumor immune microenvironment (TIME) and serves as a promising target for CRC immunotherapy.

Methods

The correlation of CD43 expression with CRC patient prognosis was revealed by public data analysis. CD43 knockout (KO) CRC cell lines were generated by CRISPR-Cas9 technology, and a syngenetic murine CRC model was established to investigate the in vivo function of CD43. The TIME was analyzed via immunohistochemical staining, flow cytometry and RNA-seq. Immune functions were investigated by depletion of immune subsets in vivo and T-cell functional assays in vitro, including T-cell priming, cytotoxicity, and chemotaxis experiments.

Results

In this study, we found that high expression of CD43 was correlated with poor survival of CRC patients and the limited infiltration of CD8+ T cells in human CRC tissues. Importantly, CD43 expressed on tumor cells, rather than host cells, promoted tumor progression in a syngeneic tumor model. Loss of CD43 facilitated the infiltration of immune cells and immunological memory in the TIME of CRC tumors. Mechanistically, the protumor effect of CD43 depends on T cells, thereby attenuating T-cell-mediated cytotoxicity and cDC1-mediated antigen-specific T-cell activation. Moreover, targeting CD43 synergistically improved PD-L1 blockade immunotherapy for CRC.

Conclusion

Our findings revealed that targeting tumor-intrinsic CD43 could activate the antitumor immune response and provide particular value for optimized cancer immunotherapy by regulating the TIME in CRC patients.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13402-023-00794-w.

Keywords: Colorectal cancer (CRC), CD43, Immunotherapy, Antitumor immune response, T cell

Introduction

Colorectal cancer (CRC), as one of the most common gastrointestinal malignancies worldwide, has increased rapidly in incidence and mortality in recent years [1]. According to the latest cancer statistics in 2022, CRC accounted for 8% of all cancers, ranking third in estimated cases and deaths [2]. CRC is characterized by concealed pathogenesis, rapid progression and poor prognosis. More than 50% of patients are diagnosed at an advanced stage (III or IV) accompanied by distant metastasis, in which the 5-year survival rate drops to less than 10% [3]. Despite significant improvements made in chemotherapy for CRC treatment, the overall survival of CRC patients remains low due to side effects, drug resistance and high recurrence [3]. For tumor immunotherapy, only 14% of CRC patients with microsatellite instability respond to PD-1 blockade therapy [4]. Thus, there persists a compelling need to discover new targets and mechanisms for early detection, diagnosis and optimized therapy of CRC. Recent studies have demonstrated that aberrant O-glycosylation modification is a crucial element of malignant phenotypes, such as proliferation, adhesion, immune invasion, metastasis and drug resistance, thus regulating the initiation and development of CRC [5–9]. However, the exact function of these glycosylated antigens in CRC and how they regulate immune surveillance remain unclear.

CD43 (also called sialophorin, SPN) is a heavily glycosylated transmembrane protein expressed on various hemopoietic cells except erythrocytes [10–14]. It has been reported to function as a T-cell counterreceptor for ICAM-1 (intercellular adhesion molecule type 1) [15, 16], E-selectin [17, 18] and galectin-1 [19] to modulate exosome secretion, T-cell adhesion, migration and exhaustion. Despite the elaborate function of CD43 on immune cells [20–28], the role of CD43 in oncology remains elusive. It has been reported that CD43 is upregulated in hematopoietic tumors [29] and correlates with worse survival outcomes in chronic lymphocytic leukemia (CLL) [30] and diffuse large B-cell lymphoma (DLBCL) patients [31]. Moreover, CD43 in human hematological malignancies has been regarded as a ligand for immune adhesion receptors, including Siglec-1 [32] and Siglec-7 [33], which bind to the α-2,3 and α-2,6-sialic acid glycoforms of CD43 respectively. Several functional assays have verified that the glycosylation of CD43 is associated with immune escape: (a) knockdown or blockade of CD43 on K562 leukemia cells could alleviate Siglec-7-mediated inhibition of natural killer-mediated cytotoxicity in vitro [33]; (b) the administration of an anti-human CD43 antibody (UN1 mAb) could inhibit the tumor growth of nu/nu mice inoculated with HPB-ALL lymphoblastoids in vivo [34]; and (c) sialic acid-rich CD43 on leukemia cells could resist CTL-mediated cytolysis in vitro and preferentially survive in wild-type mice in vivo [35].

Notably, an increasing number of publications have reported the abnormal expression of CD43 outside hematopoietic cell lineages. Some have confirmed that CD43 is expressed on the human colon carcinoma cell line COLO 205 at the protein and mRNA levels [36–38]. Subsequently, CD43 was detected in various solid tumors and cancer cell lines, including breast, cervix, thyroid, lung, pancreas and colon cancers [39–43]. One possible reason for CD43 involvement in cancer transformation is that CD43 signaling could activate Wnt/β-catenin signaling and Akt-dependent Merlin degradation [44, 45]. Strikingly, RNA interference in endogenous CD43 in human lung, cervix and colon cancer cells impaired tumor growth in nu/nu mice in vivo [45]. Furthermore, overexpression of CD43 could enhance the anti-adhesive ability of tumor cells through the modulation of integrins and extracellular matrix [46–48]. The above findings indicated that CD43 expression is critical for tumorigenesis, but whether CD43 is involved in the regulation of the tumor immune microenvironment remains unclear.

Herein, we found that CD43 expression was inversely correlated with the survival of CRC patients and the infiltration of CD8+ T cells in the CRC tissues. Importantly, we underscored that CD43 expressed on tumor cells, rather than host cells, promoted tumor progression in a syngeneic CRC model. CD43 deficiency facilitated the infiltration of immune cells and immunological memory in the TIME, and consistently, depletion of T cells abolished CD43 deletion-mediated antitumor effects. Mechanistical studies further revealed that CD43 deficiency enhanced T-cell-mediated cytotoxicity and antigen-specific T-cell activation. Interestingly, CD43-deficient CRC tumors were more prone to PD-L1 blockade immunotherapy. Hence, CD43 facilitates CRC progression by suppressing antitumor immune responses, and targeting CD43 holds promise in optimizing tumor immunotherapy regimens for CRC.

Materials and methods

Mice

All mouse experiments were conducted in accordance with the Institutional Animal Care and Use Committee of Sun Yat-sen University. C57BL/6J wild-type mice were purchased from GemPharmatech Co., Ltd. (Nanjing, Jiangsu, China). Spn−/− mice were obtained from GemPharmatech Co., Ltd., and generated by deletion of exons 1 to 2 in C57BL/6J embryonic stem cells, followed by backcrossing with C57BL/6J wild-type mice for 10 generations to construct CD43 knockout mice. Batf3−/− mice were kindly provided by Prof. Cliff Y. Yang (Zhongshan School of Medicine, China). Six- to eight-week-old mice were used in this study and housed under pathogen-free conditions in the Laboratory Animal Center of Sun Yat-sen University.

Cell lines and culture conditions

The murine colon adenocarcinoma cell line MC38 was kindly provided by Prof. Ruihua Xu’s laboratory (Sun Yat-sen University Cancer Center, China). The murine pancreatic epithelial cell line Panc02 was obtained from Prof. Xingwang Zhou (Zhongshan School of Medicine, China). All of the cell lines were cultured in DMEM (Thermo Scientific Fisher) containing 10% FBS (TransGen Biotech) and 1% penicillin-streptomycin (Thermo Scientific Fisher) at 37 ℃ in a humidified atmosphere with 5% CO2.

Mouse BMDMs were prepared as previously described by Zhong C et al. [49]. Briefly, after lysis with ACK lysis buffer (Sangon Biotech), murine bone marrow cells isolated from C57BL/6J wild-type mice were cultured in DMEM medium plus 10% FBS, 1% penicillin-streptomycin and 30% (vol/vol) L929 as CSF-1 (colony-stimulating factor 1)-conditioned medium, which was replaced with fresh medium on Days 4 and 7. Alternatively, bone marrow cells were differentiated into dendritic cells in RPMI medium (Thermo Scientific Fisher) plus 10% FBS, 1% penicillin-streptomycin, 20 ng/ml GM-CSF and 5 ng/ml IL-4 and replaced with fresh medium on Days 3 and 6.

DNA constructs and lentiviral infection

MC38 and Panc02 cells lacking CD43 were generated through CRISPR/Cas9-mediated genome editing. Briefly, the E-CRISPR tool [50] (http://www.e-crisp.org/E-CRISP/designcrispr/) was used to design the following guide RNAs: 5’-CACCGGGACCCAGCATGCCCCAAAG-3’ and 5’-CACCGCTTTGGGGCATGCTGGGTCC-3’. Subsequently, guide RNAs were cloned into the plasmid PX458 (Addgene). Cell lines were transfected with those constructs using Lipofectamine 3000 (Thermo Fisher Scientific) and were sorted for GFP expression through FACS. Polyclonal populations negative for CD43 protein were purified by 2nd sorting.

To generate MC38 derivatives stably expressing OVA, an OVA-encoding cDNA was cloned into the lentiviral vector pBOBI. Vec and CD43 KO MC38 cells were infected with mixed plasmids (pBOBI-OVA-GFP: pLP1: pLP2 = 4:3:1) using Lipofectamine 3000 and were sorted for equal GFP expression through FACS.

To rescue CD43 constructs, the full-length, extracellular or intracellular domain of CD43 was cloned and fused to pFB-Neo, a retroviral vector. MC38 CD43 KO cell lines were respectively infected with these plasmids (pFB-Neo-GFP: pCL-Eco = 1:1) using Lipofectamine 3000 and sorted according to related GFP and CD43 expression.

In vivo subcutaneous tumor transplantation assays

MC38 and Panc02 cell lines were digested into single cells using trypsin (Gibco) and washed with PBS 3 times. A total of 1 × 106 MC38, 2 × 106 Panc02 Vec or CD43 KO cell lines were inoculated subcutaneously into the right flank of 6- to 8-week-old C57BL/6J mice. From Day 6, tumor volumes were measured by calipers every 3 days and calculated using the following formula: volume=(length×width×height)/2. Mice were humanely euthanized when the tumor volume reached 1000 mm3 and recorded as death for survival curves. For the MC38 rechallenge tumor model, tumor-free mice inoculated with MC38 CD43 KO cells were rechallenged with 5 × 106 MC38 cells on Day 60.

For the depletion of immune cells, 50 µg of anti-mouse CD4 (BioXcell, GK1.5) and anti-mouse CD8α (BioXcell, 2.43) antibodies were delivered by intraperitoneal injection every week; 50 µg of anti-mouse NK1.1 (BioXcell, PK136) antibodies were injected every 3 days from Day 0; 200 µg of anti-mouse CSF1R (BioXcell, AFS98) and anti-mouse Gr-1 (BioXcell, RB6-8C5) antibodies were injected every 4 days from Day 9; and 25 µg of FTY720 inhibitors (Selleck) were administered per mouse via i.p. injection every 2 days from Day 10.

For combined therapy, mice were injected intraperitoneally with anti-mouse PD-L1 (BioXcell, 10 F.9G2) blocking antibodies at a dosage of 50 µg per mouse on Days 9, 12, 15, and 18.

Cell proliferation, colony formation and apoptosis assays

For cell proliferation, 4000 Vec or CD43 KO MC38 cells were seeded in a 96-well plate and cultured for 24, 48, or 72 h. Ten microliters of CCK-8 (DOJINDO) solution were added to each well and incubated for 4 h. Cell proliferation was determined based on the absorbance at 450 nm with a microplate reader.

For colony formation, 200 cells were plated in 6-well plates and cultured for 10 days. After fixation with 10% formaldehyde for 20 min, colonies were stained with 4.0% crystal violet for 20 min and counted by ImageJ software.

Apoptosis was detected at 48 h using the Annexin V/PI kit (Biolegend). Early and late apoptosis were considered as Annexin V+/PI− and Annexin V+/PI+ populations, respectively.

Flow cytometry analysis

Tumor tissues were minced and digested by HBSS buffer (Sangon Biotech) containing 10% FBS, 1% penicillin-streptomycin, 10 mM HEPES, 50 µg/ml DNase I, 1 mg/ml dispase II, 1.25 mg/ml collagenase D and 0.85 mg/ml collagenase V (Sigma-Aldrich), at 80-rpm speed at 37 °C for 40 min. The digested cells were filtered through 70-µm cell strainers (Millipore) and centrifuged at 600 g and 4 °C for 5 min. Then, 5 × 106 cells were counted and resuspended in 100 µl of FACS buffer (PBS plus 2% FBS). Subsequently, 0.2 µg of anti-mouse IgG2a (BioXcell, C1.18.4) and CD16/32 (BioXcell, 2.4G2) antibodies were added to block FcR for 30 min on ice. The cells were stained with 0.4 µg of specific antibodies for 30 min on ice. 7-AAD viability staining solution, anti-mouse FITC-conjugated-CD11b (M1/70), PE-conjugated-CD11c (N418), PE-conjugated-CD39 (Duha59), PE/Cyanine-conjugated-F4/80 (BM8), PE/Cyanine7-conjugated-CD8 (53 − 6.7), APC-conjugated-Ly6G (1A8), APC-conjugated-CD43 (S11), APC-conjugated-CD3 (145-2C11), Alexa Fluor 700-conjugated CD45 (30-F11), APC/Cyanine7-conjugated-I-A/I-E (M5/114.15.2), Brilliant Violet 421-conjugated-Ly6C (HK1.4), Brilliant Violet 421-conjugated-NK1.1 (PK136), Brilliant Violet 510-conjugated-CD80 (16-10A1), Brilliant Violet 510-conjugated-PD-1 (29 F.1A12), Brilliant Violet 605-conjugated-anti-PD-L1 (10 F.9G2), Brilliant Violet 605-conjugated-CD4 (RM4-5) and Brilliant Violet 650-conjugated-CD19 (6D5) antibodies were purchased from BioLegend. Flow cytometry was performed with a Cytoflex analyzer (Beckman Coulter) and analyzed with FlowJo software.

Immunohistochemistry

Dissected tumor tissues were fixed with 4% formaldehyde and stained with hematoxylin and eosin (H&E) by Servicebio company. Then the infiltration of CD45+ and CD3+ cells was visualized by IHC staining and calculated with a 20× objective using ImageJ software.

RNA-seq

Total RNA of bulk tumors was extracted with TRIzol reagent (Sigma-Aldrich) according to the given instructions. One microgram of RNA per sample was used as the input for the RNA sample preparations. Sequencing libraries were generated using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA), with index codes added to attribute sequences to each sample. For RNA-seq analysis, following standard methods, raw reads were processed, and differentially expressed genes were quantified and identified using DEseq2. Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the clusterProfiler R package, which corrected gene length bias. GO terms with corrected p values less than 0.05 were considered significantly enriched, differentially expressed genes. KEGG is a database resource for comprehending high-level functions and applications of the biological system (http://www.genome.jp/kegg/). The ClusterProfiler R package was utilized to examine the statistical enrichment of annotated differentially expressed genes in KEGG pathways.

ELISA

Mouse interferon-γ (Servicebio, EK280/3 − 01) and interleukin-2 (Servicebio, EK202/2 − 01) ELISA kits were used to determine the concentrations of interferon-γ and interleukin-2 in tumor interstitial fluids following standard protocols.

In vitro phagocytosis assay

Phagocytosis assays were performed according to the previous articles [51, 52]. BMDMs (5 × 104) were seeded overnight in a 24-well tissue culture plate. The next day, 2 × 105 Vec or CD43 L1210 target cells were labeled with 2.5 µM of CFSE (Thermo Scientific Fisher) and incubated with macrophages in non-FBS medium plus 10 µg/ml anti-mouse CD47 mAb or control IgG for 2 h at 37 °C. Then, macrophages were washed with PBS for 3 times and imaged with a fluorescence microscope. Percent phagocytosis was calculated using the following equation: (the number of macrophages engulfing CFSE+ target cells) /total macrophages×100.

In vitro T-cell functional assays

For the T-cell cytotoxicity assay, 5 × 105 CD8+ T cells derived from splenocytes of OT-I transgenic mice were enriched with the EasySep™ Mouse CD8+ T-Cell Isolation Kit (Stemcell). A total of 1 × 105 Vec or CD43 KO MC38-OVA-GFP cells were resuspended in the complete medium containing 10% FBS, 20 ng/ml IL-2 (Peprotech), and 50 µM β-mercaptoethanol (Sigma-Aldrich) and added to the effector cells in a 24-well plate. After 36 h of incubation, apoptotic tumors were analyzed with flow cytometry in line with negative GFP expression.

For the T-cell priming assay, BMDCs and Vec or CD43 KO MC38-OVA-GFP cells were incubated in 6 cm dishes at a ratio of 1:3. After 6 h, the coculture supernatants were collected and BMDCs were sorted through FACS. Then, 1 × 105 OVA-loaded BMDCs were incubated with 4 × 105 CFSE-labeled OT-I CD8+ T cells in the mixed media containing 200 µl of supernatants and 400 µl of T-cell conditioned medium (as mentioned above) for 48 h and then subjected to FACS analysis to determine the CFSE intensity of CD8+ T cells.

For the T-cell recruitment assay, BMDCs and Vec or CD43 KO MC38-OVA-GFP cells were seeded in 24-well flat-bottom plates. A total of 4 × 106 splenocytes isolated from OT-I transgenic mice were placed in the upper chamber (8 μm, BD Falcon). After 2 days, cells in the bottom wells were collected and stained with anti-mouse APC-conjugated-CD3 (145-2C11), Brilliant Violet 605-conjugated-CD4 (RM4-5) and PE/Cyanine7-conjugated-CD8 (53 − 6.7) antibodies for 30 min on ice. Then, the number of migratory T cells was quantified with flow cytometry and analyzed with FlowJo software.

Gene expression data and clinical data acquisition for colon cancer

The expression profile and clinical data of colon cancer and adjacent normal tissues from The Cancer Genome Atlas (TCGA) along with normal colon tissues from Genotype-Tissue Expression (GTEx) were downloaded from XENA (http://xena.ucsc.edu/). These two datasets were transformed into TPM data and then normalized and combined in terms of the description of relevant cohorts. Expression and survival data of colon cancer were also extracted from the GEO database (https://www.ncbi.nlm.nih.gov/geo/), including GSE38832 (n = 92), GSE72968 (n = 68), and GSE72969 (n = 56). These expression profiles from GEO were preprocessed by RMA with the affy R package, and normalized expression data were downloaded for further analysis.

Correlation analysis between CD43 expression and the abundance of immune cells

Based on expression profiles from colon cancer patients in TCGA, the abundance of each type of tumor-infiltrating immune cells was calculated by CIBERSORT (PMID: 25,822,800) and xCell (PMID: 29,141,660). Immune cell infiltration data were joined with the corresponding expression level of CD43, with which Pearson’s correlation analyses were performed, and correlation graphs were constructed via ggplot2 in R.

Kaplan-Meier survival analysis

With regard to the above three colon cancer GEO cohorts, the best separations of high and low CD43 expression were computed through the survminer package in R. Subsequently, Kaplan-Meier survival curves were plotted by the survival package.

Statistical analysis

GraphPad Prism software, version 8.0, was used for statistical analysis and graphing. In all cases, the data are presented as the mean ± SEM from independent biological replicates. Statistical significance was determined by Student’s unpaired t test (two-tailed) between two groups, labeled * p < 0.05, ** p < 0.01, *** p < 0.001. **** p < 0.0001. The survival curves were analyzed with the log-rank test.

Results

High expression of CD43 in colorectal cancer correlates with worse survival outcomes

The expression level of CD43 was significantly increased (p < 0.001) in 290 colon cancer tissues compared with 349 normal colon samples in the TCGA and GTEx cohorts (Fig. 1a). Two immune cell quantification algorithms, CIBERSORT and xCell, were applied to analyze the correlation between the expression of CD43 and the infiltration of colon cancer immune cells. The expression level of CD43 was significantly negatively correlated with the abundance of effector memory CD8+ T cells (Fig. 1b) and NK cells (Fig. 1c), whereas a positive correlation was demonstrated with the infiltration of Tregs (Fig. 1d). To further investigate the role of CD43 in colon cancer survival, samples from several datasets were divided into high and low expression groups under the optimal threshold. CRC patients with low CD43 expression were significantly associated with better DFS, OS, and PFS (p < 0.05) than those with high expression (Fig. 1e-h). Collectively, CD43 was highly expressed in tumor tissues, indicating poor prognosis in colon cancer patients.

Fig. 1.

Fig. 1

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High expression of CD43 in colorectal cancer correlates with worse survival outcomes. (a) Expression levels of CD43 in CRC and adjacent normal colon samples in the TCGA and GTEx cohorts. (b-d). Correlation analysis between the expression of CD43 and the infiltration of immune cells in CRC: (b) effector memory CD8+ T cells, (c) NK cells, and (d) Tregs. (e-h). Kaplan-Meier curves of (e) DFS in GSE38832, (f) OS in GSE72968, PFS in (g) GSE72968 and (h) GSE72969 cohorts, comparing high and low CD43 expression. DFS, disease-free survival time; OS, overall survival time; PFS, progression-free survival time

CD43 protects solid tumor development

To reveal the potential role of CD43 in solid tumor development, we generated the CD43-deficient colon cancer cell line MC38 by CRISPR-Cas9 technology (Fig. 2a, Fig. S1a-left). Intriguingly, CD43 KO MC38 tumors progressed significantly slower (Fig. 2b), with dramatically smaller tumor sizes (Fig. 2c) and tumor weights (Fig. 2d). Furthermore, much longer overall survival was observed in CD43 KO MC38 tumor-inoculated mice than in control mice (Fig. 2e). To confirm this discovery, we then generated the CD43-deficient pancreatic tumor cell line Panc02 (Fig. 2f, Fig. S1a-right). Consistently, CD43 KO Panc02 tumors were obviously smaller than control tumors after inoculation in mice in vivo (Fig. 2g). Since CD43 was highly expressed in colon cancer (Fig. 1a), we wondered whether CD43 could regulate the proliferation or apoptosis of tumor cells. However, similar growth rates were observed between CD43 KO and control tumor cells (Fig. S1b). Colony formation assays also revealed no obvious difference in CD43 KO or control tumor cells (Fig. S1c, d). Finally, very mild apoptosis was detected in both CD43 KO tumor cells and control tumor cells (Fig. S1e, f).

Fig. 2.

Fig. 2

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CD43 protects solid tumor development. (a) Expression level of CD43 on Vec (the blue line) and CD43 KO (the red line) MC38 cells detected by FACS after CRISPR/Cas9-mediated gene knockout. (b-e) (b) Tumor volume, (c) representative image of xenograft tumors and (d) corresponding tumor weight (n = 5), and (e) survival curve of Vec or CD43 KO MC38-bearing wild-type mice. (f) Expression level of CD43 in Vec (the green line) and CD43 KO (the pink line) Panc02 cell lines. (g) Tumor volume of Vec and CD43 KO Panc02-bearing mice. (h) Tumor volume of MC38-bearing WT or Spn−/− mice. (i) Schematic representation of full-length (FL) CD43, along with the indicated extracellular (ED) and intracellular (ID) truncations. (j) Subcutaneous tumor volume and (k) survival curve of mice inoculated with Vec (n = 9), KO (n = 10), Vec-FL (n = 4), KO-FL (n = 10), KO-ED (n = 10) and KO-ID (n = 9) MC38 cell lines. The results are labeled as the mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s., not significant

Importantly, CD43 is highly expressed in immune cells, especially in T cells [10–13]. To test whether the CD43 expression in host immune cells was critical, we generated CD43-deficient mice. However, MC38 tumors in CD43 KO mice developed quite similarly to those in WT mice (Fig. 2h), suggesting that CD43 in host cells is negligible for MC38 tumor progression. We then designed a series of CD43 mutant constructs to determine the functional domain of CD43 expression on MC38 cells (Fig. 2i, Fig. S1g). The rescued full-length CD43 restored the defective tumor growth in CD43 KO tumors (Fig. 2j, k). Interestingly, the reconstituted intracellular domain of CD43 (KO-ID) could largely restore the suppressed tumor growth in CD43 KO tumors, whereas the reconstituted extracellular domain of CD43 (KO-ED) showed a weak effect (Fig. 2j, k), in line with a previous report that the intracellular domain is critical for CD43 function on T cells [27, 28]. These data indicated that CD43 expressed on tumor cells supported colon cancer progression, which occurred not through regulating the intrinsic activity of tumor cells.

CD43 deficiency facilitates immune cell infiltration in the TIME

We then speculated that CD43 might regulate the tumor microenvironment in vivo. CD43 KO or control MC38 tumors were collected from mice at 18 days postinoculation and subjected to immunohistochemical staining. In the control tumors, few CD45+ positive immune cells (Fig. 3a-top) and CD3+ T cells (Fig. 3c-top) were detected, while most of the immune cells localized at the marginal regions of tumors. Intriguingly, CD43 deficiency dramatically facilitated the infiltration of CD45+ positive immune cells (Fig. 3a-bottom, 3b) and CD3+ T cells (Fig. 3c-bottom, 3d) into the TIME, and importantly, more immune cells penetrated into the central regions of the CD43 KO tumors more than in the control tumors (Fig. 3a, c). Next, the TIME was analyzed by flow cytometry. Infiltration of CD45+ immune cells was significantly increased in CD43 KO tumors compared to control tumors (Fig. 3e, f), consistent with the results of immunohistochemical staining (Fig. 3a, b). Although the total myeloid immune populations (CD11b+ cells) (Fig. S2a), macrophages (Fig. S2b), DCs (Fig. S2c) and P-MDSCs (Fig. S2d) were similar between CD43 KO and control tumors, Ly6Chi cells were dramatically enriched in CD43 KO tumors (Fig. 3g). For lymphocyte populations, significantly more NK cells (Fig. 3h), T cells (CD3+ cells) (Fig. 3i), and CD8+ T cells (Fig. 3j) were observed in CD43 KO tumors than in control tumors, while B cells (Fig. S2e) and CD4+ T cells (Fig. S2f) remained at similar levels. Interestingly, CD43 KO tumors recruited more PD-1hiCD39+CD8+ T cells (Fig. 3k), which were reported to function as tumor antigen-specific effector T cells in tumors [53]. Collectively, these results revealed that CD43 deficiency switched ‘cold tumors’ to ‘hot tumors’, which was especially shown by sufficient recruitment of T cells into the TIME.

Fig. 3.

Fig. 3

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CD43 deficiency facilitates immune cell infiltration in the TIME. (a-d) Representative immunohistochemistry and AOD (area of density) statistics for (a and b) CD45 and (c and d) CD3 staining of Vec and CD43 KO MC38 tumors at 18 days postinoculation (n = 4). (e) Representative FACS plot of infiltrating CD45+ immune cells in Vec and CD43 KO xenograft tumors on Day 18 postinoculation (gating on 7AAD− populations). (f-k) Frequencies of (f) CD45+ cells, (g) Ly6Chigh neutrophils, (h) NK cells, (i) T cells, (j) CD8+ T cells and (k) PD-1hi CD39+ CD8+ T cells in the TIME measured by flow cytometry (n = 6). The results are displayed as the mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Deletion of CD43 enhances T-cell activation

To elucidate the TIME more precisely, tumors were harvested at Day 21 postinoculation in vivo, and single-cell suspensions of tumor tissues were generated for transcriptome sequencing (Fig. 4a). We found abundant differentially expressed genes between control and CD43 KO tumor tissues (Fig. 4b). The significantly enriched gene ontology (GO) terms in upregulated genes of CD43 KO tumors included T-cell activation, cytokine production, activation of immune response and leukocyte chemotaxis, which favor antitumor immune response (Fig. 4b, c). Consistently, the cytokine-cytokine receptor interaction, T-cell receptor signaling pathway, cytotoxicity, and chemokine signaling pathway were enriched in the KEGG term analysis of CD43 KO tumors (Fig. 4d).

Fig. 4.

Fig. 4

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Deletion of CD43 enhances T-cell activation. (a) Workflow of transcriptome analysis for bulk tumors on Day 21 after inoculation. (b) Volcano plot (thresholds: at least 2-fold change, p < 0.05), (c) GO and (d) KEGG analysis of differentially expressed genes between the Vec (n = 4) and CD43 KO (n = 5) groups. (e-f) ELISA for the concentration of (e) interferon-γ and (f) interleukin-2 in tumor interstitial fluids between the Vec (n = 6) and CD43 KO (n = 5) groups. (g) Representative flow cytometric plots and (h) quantification of the cytotoxicity of OT-I CD8+ T cells pulsed with Vec or CD43 KO MC38-OVA-GFP cells for 36 h (from three independent experiments). (i-j) Tumor volumes of Vec and CD43 KO MC38-bearing mice treated with (i) anti-CD8 and (j) anti-CD4 antibodies. The results are depicted as the mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001

These results were further verified by experimental approaches. First, ex vivo homogenized tumor interstitial fluids were analyzed by ELISA, revealing that IFN-γ (Fig. 4e) and IL-2 (Fig. 4f) were upregulated in CD43 KO tumor tissues. Moreover, we established a T-cell cytotoxicity assay by incubating Vec or CD43 KO MC38 cells expressing OVA peptide with OVA peptide-specifically reactive OT-I T cells for 36 h (Fig. S3a). By detecting apoptosis of MC38 cells with flow cytometry, significantly more CD43 KO tumor cells were killed by OT-I T cells than control tumor cells (Fig. 4g, h). Therefore, this evidence demonstrated that CD43 deficiency facilitates T-cell activation and functions. We then wanted to determine the contribution of T cells to CD43 deficiency-induced antitumor immunity. Intriguingly, CD43 KO tumors rapidly grew to sizes similar to those of control tumors when CD8+ T cells were depleted by specific antibodies (Fig. 4i), while CD4+ T-cell depletion also partially rescued the retarded tumor volumes in CD43 KO tumor-inoculated mice (Fig. 4j). However, deletion of NK cells, another type of immune effector cells, barely affected CD43 KO tumor sizes (Fig. S3b). Therefore, CD43 expression protected tumor cells from T-cell-mediated cytotoxicity, and targeting CD43 facilitated robust T-cell activation.

CD43 deficiency renders effective immunological memory

Antigen-specific T cells prime, activate, and proliferate in tumor-draining lymph nodes (TDLNs), followed by the recruitment of effector T cells into tumor tissues by chemotaxis originating from the TIME. Interestingly, we found significantly increased cDCs (Fig. 5a) and CD8+ T cells (Fig. 5b) in TDLNs from CD43 KO tumor-inoculated mice compared with those from control tumors, while the CD45+ immune cells (Fig. S4a), CD11b+ myeloid cells (Fig. S4b), macrophages (Fig. S4c), DCs (Fig. S4d), NK cells (Fig. S4e), B cells (Fig. S4f), T cells (Fig. S4g) and CD4+ T cells (Fig. S4h) were similar between TDLNs from CD43 KO and control tumor-bearing mice. S1P signaling is critical for T-cell migration from TDLNs, and its inhibition greatly reduces the antigen-specific T cells in the peripheral organs and also in the tumor tissues [54]. Notably, CD43 KO tumors were dramatically larger after FTY720 (an S1P inhibitor) treatment than nontreated CD43 KO tumors (Fig. 5c), indicating that CD43 deficiency-mediated antitumor immunity required the antigen-specific T cells in TDLNs.

Fig. 5.

Fig. 5

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CD43 deficiency renders effective immunological memory. (a-b) Frequencies of (a) cDCs and (b) CD8+ T cells in TDLNs from Vec and CD43 KO tumor-inoculated mice on Day 21 (n = 4). (c) Tumor volume of Vec and CD43 KO MC38-bearing mice with S1P inhibitor (FTY720) treatment. (d) Tumor volume of WT or Batf3−/− mice inoculated with Vec and CD43 KO MC38 cells. (e) Timeline of the MC38 rechallenge model. (f) Tumor volume and (g) survival curve of 8-week-old WT control or related CD43 KO-primed mice rechallenged with 5-fold primary injected MC38 cell lines. The results are expressed as the mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001

Previous studies have reported that antigen-presenting cells, including DCs and macrophages, play critical roles in antigen-specific T-cell differentiation and proliferation [55, 56]. Interestingly, CD43 deficiency-mediated tumor control was dramatically attenuated in Batf3 KO mice (Fig. 5d), which lacked the cDC1 population. However, macrophage depletion by anti-CSFR1 antibodies (Fig. S4i) or depletion of neutrophils by anti-Gr-1 antibodies (Fig. S4j) had a mild effect on CD43 KO tumor development. Moreover, macrophages phagocytosed CD43 KO tumor cells or control tumor cells at similar levels (Fig. S4k, l), suggesting that CD43 targeting primarily enhanced cDC1 function rather than macrophages or neutrophils. Therefore, the immune crosstalk between cDC1 and T cells was reinvigorated by CD43 targeting, which was speculated to induce immunological memory. We observed that some CD43 KO MC38 tumors spontaneously vanished, and these convalescent mice were rechallenged with 5-fold numbers of the primary injected tumor cells (Fig. 5e). The rechallenged tumors developed rapidly in control mice (Fig. 5f), which were humanely sacrificed by 30 days postinoculation (Fig. 5g). Crucially, the rechallenged tumors were absolutely rejected in convalescent mice (Fig. 5f) that were alive until Day 70 post-rechallenge (Fig. 5g). Thus, CD43 targeting reinvigorated cDC1 and T-cell activation in TDLNs, which triggered efficient immunological memory against tumors.

Deletion of CD43 facilitates antigen-specific T-cell activation

We then wanted to explore the mechanism by which CD43 regulates antigen-specific T-cells activation by DCs. First, OVA-expressing control or CD43 KO tumor cells were cocultured with BMDCs in the non-FBS medium, and consequently, tumor-pulsed BMDCs were purified by flow cytometry sorting and incubated with CFSE-labeled OT-I CD8+ T cells for 48 h at the indicated ratio. Finally, these CD8+ T cells were subjected to FACS analysis to determine the proliferation of CD8+ T cells (Fig. 6a). However, under these conditions, control or CD43 KO tumor cell-primed BMDCs induced equivalent T-cell proliferation (Fig. 6b, c). Considering that the CD43 KO TIME was enriched in cytokine and chemokine signaling (Fig. 4b-d), and CD43 was reported to mediate the interaction between DCs and T cells through a nonclassical pathway of secretion via exosomes, we speculated that these soluble factors might be critical for T-cell proliferation. Crucially, supplementation with 1/3 tumor-BMDC coculture supernatants into T-cell conditioned medium (Fig. 6d), enabled more efficient T-cell proliferation by CD43 KO tumor cell-primed BMDCs compared with the control group (Fig. 6e, f). The CD43-deficient TIME highly secreted many cytokines and chemokines, as revealed by RNA-seq data (Fig. 6g), and evidenced by the increased recruitment of T cells by CD43 KO tumor cell-primed BMDCs (Fig. 6h, i). Therefore, CD43 deficiency in tumors promotes DCs motivation and antigen-specific T-cell activation and recruitment.

Fig. 6.

Fig. 6

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Deletion of CD43 facilitates antigen-specific T-cell activation. (a) Workflow, (b) flow cytometric plots and (c) statistics of priming of antigen-specific CD8+ T cells incubated with OVA-loaded BMDCs (n = 3). (d) Workflow, (e) FACS images and (f) quantification of the proliferation of CD8+ T cells incubated with pretreated BMDCs plus collected coculture supernatants (n = 3). (g) Heatmaps of cytokine and chemokine-related genes of CD43 KO versus Vec based on expression values. (h) Schematic and (i) statistics of migratory T cells recruited by BMDCs cocultured with Vec or CD43 KO MC38 OVA-GFP cells (n = 5). The results are presented as the mean ± SEM, *** p < 0.001, **** p < 0.0001

Targeting CD43 optimizes immune checkpoint blockade (ICB) tumor immunotherapy

We found abundant differentially expressed genes between control and CD43 KO tumor tissues by RNA-seq analysis (Fig. 4b). PD-1 and PD-L1 immune checkpoint signaling-related genes were significantly enriched in CD43 KO tumors by GSEA (Fig. 7a, b), indicating that CD43 deficiency could potentially synergistically improve ICB immunotherapy. Interestingly, loss of CD43 in tumors suppressed tumor growth comparable with anti-PD-L1 antibody treatment, while CD43 KO tumors were much more sensitive to anti-PD-L1 antibody treatment than control tumors (Fig. 7c). Intriguingly, the combination of CD43 deficiency and PD-L1 blockade led to as high as 50% curation of tumor-bearing mice. However, either CD43 deficiency or PD-L1 blockade only rendered approximately 20% curation of tumor-inoculated mice (Fig. 7d). These data suggested that CD43 targeting could serve as a priming strategy to optimize current cancer immunotherapy for CRC.

Fig. 7.

Fig. 7

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Targeting CD43 optimizes immune checkpoint blockade (ICB) tumor immunotherapy. (a-b) GSEA of differentially expressed genes between the Vec (n = 4) and CD43 KO (n = 5) groups. (c) Tumor volume and (d) survival curve of Vec or CD43 KO MC38-bearing mice administered intraperitoneally with anti-mouse PD-L1 or left untreated on Days 9, 12, 15 and 18. The results are labeled as the mean ± SEM, * p < 0.05, ** p < 0.01, n.s., not significant

Discussion

CRC is one of the most life-threatening cancers worldwide. Although ICB immunotherapies present positive clinical outcomes for a proportion of CRC patients diagnosed with microsatellite instability (MSI), the overall benefits of current immunotherapy for CRC treatment remain very limited [4]. Dedicated efforts are required to explore novel potential mechanisms and targets for CRC immunotherapies.

CD43, a mucin-like type I transmembrane protein expressed on immune cells (e.g., T cells), hematological malignancies and some solid tumors, was reported to regulate immune signaling transduction, tumor proliferation and tumorigenesis [57]. In this study, we found that high expression of CD43 was correlated with poor clinical outcomes of CRC (Fig. 1), while CD43 deficiency did not affect tumor-intrinsic survival or proliferation (Fig. S1b-f). Intriguingly, loss of CD43 in the CRC cell line and pancreatic cell line significantly attenuated tumor development in syngeneic murine cancer models (Fig. 2b-e, g). Although CD43 was reported to function on immune cells [20–26], MC38 CRC tumors developed similarly in CD43 KO mice (Fig. 2h). These findings indicated that CD43 expressed on tumor cells other than host cells was crucial, suggesting that CD43 expressed on different cell types could have distinct functions.

In addition, previous studies have demonstrated that the inhibitory function of CD43 depends on its intracellular domain rather than the extracellular domain with a negative charge and extended structure. In particular, phosphorylation of the cytoplasmic domain leads to its interaction with ezrin-radixin-moesin (ERM) cytoskeletal adaptor proteins, resulting in decreased IL-2 production and T-cell migration [27, 28], in accordance with our finding that the regulation of CD43 for CRC tumor development depended on its intracellular domain in vivo (Fig. 2i-k). Since the intracellular domain of murine CD43 is 70% identical to that of human CD43 [58], the high homology of the ID domain remains to be further explored to dissect its mechanism in CRC progression.

By investigating the TIME via immunohistochemical staining and flow cytometry, we discovered dramatic infiltration of immune cells in CD43 KO tumors (Fig. 3). Consistently, RNA-seq data analysis revealed obvious immune signatures, including T-cell activation, leukocyte chemotaxis and T-cell receptor signaling pathways (Fig. 4b-d), which were evidenced by the elevated IFN-γ and IL-2 production and cytotoxicity of T cells in CD43 KO tumors (Fig. 4e-h). These results strongly suggested that CD43 regulated immune infiltration and T-cell activation in the TIME, further supported by the abolished antitumor activity in CD43 KO tumors after depletion of T cells (Fig. 4i, j), and the prohibition of antigen-specific T-cell migration out of the LNs (Fig. 5c).

Furthermore, antigen-specific T-cell responses are normally primed by antigen-presenting cells (APCs) in LNs [59]. Immune subset deletion assays clarified that cDC1s, rather than macrophages, were the APCs responsible for T-cell activation in the TIME of CD43 KO tumors (Fig. 5d, S4j-l). Additionally, CD43 KO tumor-primed DCs cells induced more antigen-specific T-cell proliferation and chemotaxis (Fig. 6). Impressively, CD43 KO CRC tumor-primed mice triggered robust immunological memory against rechallenged tumors (Fig. 5f, g). These data proved that CD43 deficiency reinvigorated both innate and adaptive immune responses in CRC tumors, representing a novel mechanism of CD43.

The major obstacles for current ICB immunotherapy with limited long-term efficacy include inevitable T-cell exhaustion and the lack of de novo tumor antigen-specific T-cell activation [60]. In light of the substantial challenges of current immunotherapy strategies, novel mechanisms and targets are urgent issues for optimized immunotherapies. Our findings indicated that targeting CD43 might facilitate de novo tumor antigen-specific T cells by cDC1s in LNs (Fig. 5c, d) to compensate for the continually exhausted T cells in the TIME. This hypothesis was evidenced by that targeting CD43 synergistically potentiated the efficacy of PD-L1 immunotherapy (Fig. 7c, d). Considering these findings together, CD43 facilitates CRC progression by constraining antitumor immune responses, and targeting CD43 is expected to optimize tumor immunotherapy regimens for CRC.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (595.1KB, pdf)

Authors’ contributions

J.C. and Y.Y.L. wrote the original manuscript. Y.Y.L. and X.Y.W. conceived and conducted most of experiments and data curation. Y.L. participated in the data analysis of the clinical samples. X.M.W., J.L., Y.Z.W. and H.H. assisted with some animal experiments. J.C. and W.Y. supervised the research and authored the final manuscript. All authors reviewed the manuscript. We thank Figdraw (https://www.figdraw.com) for expert assistance in the pattern drawing.

Funding

This work was supported by National Key R&D Program of China (2020YFA0509400 and 2019YFA0110300), National Natural Science Foundation of China (82150117, 82071745 and 82101329), Science and Technology Program of Guangzhou (202002030069), Guangdong project (2019QN01Y212), Guangdong Basic and Applied Basic Research Foundation of China (2021A1515012620), Guangzhou Science and Technology Project of China (202201010993 and 105068559019) and a Grant from MOE Key Laboratory of Gene Function and Regulation.

Data Availability

The analyzed datasets generated during this study are available from the corresponding author upon reasonable request. The transcriptomic data are available in Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/) under the accession codes GSE223856.

Declarations

Competing interests

There is no conflict of interest in our article.

Ethical approval

All of the animal experiments were performed with the approval of the Institutional Animal Care and Use Committee, Sun Yat-Sen University.

Footnotes

Yi-yi Li and Xin-yu Wang contributed equally to this work.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Wei Yi, Email: yiwei@gzzoc.com.

Jun Chen, Email: chenjun23@mail.sysu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (595.1KB, pdf)

Data Availability Statement

The analyzed datasets generated during this study are available from the corresponding author upon reasonable request. The transcriptomic data are available in Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/) under the accession codes GSE223856.

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