B7 molecule mRNA expression in colorectal carcinoma

Abstract

AIM: To observe the status of tumor-associated B₇ molecule mRNA expression in human colorectal cancer tissue by in situ hybridization.

METHODS: The mRNA expression patterns of cancer-associated B_7-1,B₇H₁,B₇H₂,ICOS in 22 specimens of human colorectal cancer tissue were monitored by in situ hybridization (ISH) with digoxin-labeled oligonucleotide probes.

RESULTS: B_7-1,B₇H₁,B₇H₂,ICOS mRNA were detected in both cancer cells and tumor infiltrating lymphocytes (TIL). The mRNA expression level of these molecules in tumor cells was higher than that in TIL (0.76 ± 0.54-1.62 ± 0.82 vs 0.38 ± 0.19-0.65 ± 0.33, P<0.001). There was no relationship between expression level of tested B₇ family molecules and patients’ sex, age, differentiation status of cancer and regional lymph node metastasis.

CONCLUSION: Th2 cytokine predominant in tumor microenvironment might be related to the expression of B₇H₁,B₇H₂ co-signal molecules in tumor cells and TIL.

Key Words: Colorectal cancer, B_7-1, B7H1, B7H2 and ICOS, Tumor immunity, Immune evasion

INTRODUCTION

Although it has been well accepted that human tumor is immunogenic, most patients suffering from cancer are destined to die due to tumor progress. Establishment of the bisignal model for T-cell activation leads people to think that inactivation of infiltrating immune potential cells in tumor tissue might be immune anergic owing to the deficiency of co-stimulatory molecules. Tumor cells modified with B₇ co-stimulatory molecule gene could be rejected by tumor bearing host[1,2], but most human cancer tissues express co-stimulators[3,4], suggesting that co- stimulatory molecules might not be the only mechanism of immune evasion. The tumor-associated B₇H₁ and B₇H₂, new members of B₇ family can preferentially stimulate the production of IL-10, promote activated T cell apoptosis[5].

In the present study, the status of tumor-associated B₇ molecule mRNA expression in human colorectal cancer tissue was observed using B₇H₁ , B₇H₂ and ICOS cDNA probe, in situ hybridization. The results showed that B_7-1, B₇H₁ , B₇H₂, ICOS mRNA were expressed in both cancer cells and tumor infiltrating lymphocytes (TIL), indicating that Th2 cytokine predominant in tumor microenvironment might be related to the expression of B₇H₁, B₇H ₂ co-signal molecules in tumor cells and TIL. Elucidation of tumor-associated B₇ molecules may contribute to the design of T cell-based cancer immunotherapy.

MATERIALS AND METHODS

Samples

Tissue samples were obtained from 22 patients with colorectal cancer. The specimen were fixed in 4 g/L formaldehyde in phosphate-buffered saline (PBS) immediately and embedded in paraffin. Serial sections (5 μm in thickness) were cut for in situ hybridization or histological evaluation, and mounted on slides covered with APES, dried overnight at 65°C, stored at -70°C until use. The diagnosis of colorectal carcinoma was histopathologically verified in all cases. None of the patients had previously received radio-, chemo-, or immunotherapy.

Preparation of oligonuleotide probes

Primer 3 software was used to design oligonuleotide probes complementary to the mRNA of all kinds of target sequences including B_7-1, B₇H₁, B ₇H₂, ICOS mRNA. The specificity of all oligonucleotide probes was analyzed by BLAST software (http://www.ncbi.nih.gov). The probes were labeled by tailing the oligonucleotides with digoxigenin-11-dUTP kit (Boehringer Mannheim, German). A labeling activity of 1.56 nmol/L was obtained (Primer3 http://www. genome.wi.mit.edu/cgi-bin/primer/Primer3;BLAST http://www.ncbi.nlm.gov/cgi-bin/BLAST). Sequence of oligonuleotide probes of co-stimulatory molecules was tested (B_7-1: 5’-CAT GAA GCT GTG GTT GGT TG -3’; B₇H₁: 5’-TGC TTG TCC AGA TGA CTT CG -3’; B₇H₂: 5’-CCA TCG CTC TGA CTT CCT TC -3’; ICOS: 5’-TTC AGC TGG CAA CAA AGT TG-3’).

In situ hybridization (ISH)

Diethyl pyrocarbonate (DEPC) water was used for all solutions necessary for ISH. The sections were deparaffinized in xylene and rehydrated in descending ethanol, followed by digestion with 1 g/L proteinase K at 37°C for 30 min and terminated with 20 g/L glycine in PBS for 5 min. Then, the sections were refixed in 40 g/L polyformaldehyde for 20 min, washed with PBS for 10 min, treated with 0.2 mol/L HCI for 10 min and washed with DEPC water for 3 min. The sections were dehydrated in ascending gradient ethanol, air-dried and followed by prehybridization at 42°C for 2 h. Hybridization reaction was carried out at 42°C for 22 h. Then the sections were washed with gradient SSC thoroughly and followed by treatment with digoxin antibody at 37°C for 2 h. The color was developed in NBT-BCIP substrate, then counterstained with 10 g/L methyl green in distilled water, dehydrated and mounted with neutral gum.

For negative controls, probes and antibodies were replaced by PBS or the slides were treated with RNase A (20 mg/L) at 37°C for 30 min. B_7-1 mRNA expression in mononuclear leucocytes was used as positive control.

Evaluation of results

Purple blue precipitation in cytoplasm under light microscope was considered as positive signal, and 5 high power fields were randomly chosen from each slide, The percentage of positive cells and the positive cell index (total positive granule number/positive cell number) were calculated. Intensity of the color reaction was classified into 4 grades: strongly positive (+++), positive (++), weakly positive (+) and negative (-) and scored as 3, 2, 1 and 0, respectively. The Accumulation index was determined as percentage of positive cells multiply intensity score.

Statistics analysis

All datas were expressed as mean±SD. Analysis was performed by using chi-square test and Spearman correlation analysis with SPSS 11.0 software. P<0.05 was considered statistically significant.

RESULTS

Expression of B_7-1, B₇H₁, B₇H₂, ICOS mRNA was detected in both cancer cells and tumor infiltrating lymphocytes (Figure 1) . The mRNA expression level of these molecules in tumor cells was higher than that in TIL (0.76 ± 0.54-1.62 ± 0.82 vs 0.38 ± 0.19-0.65 ± 0.33, P = 0.000). However, when the expression of B₇ family mRNA was analyzed with respect to the tested members, there was a significant difference in B₇H₁ and ICOS expression between TIL and cancer cells (P<0.05) . The expression of B₇H₁ and ICOS mRNA was higher in tumor cells and TIL. The expression of B₇ family molecules either in tumor cells or in TIL was not correlated with patients’ sex, age, differentiation status of cancer and regional lymph node metastasis. B₇H₁ expression in TIL and tumor invasiveness was significantly associated with the intensity and the depth of tumor invasion (P = 0.050, Table 1, Table 2, Table 3).

Table 1 B7 family molecule expression in colorectal cancer cells and TIL (mean±SD).

B7 molecule	Tumor cell	TIL
B7-1:	1.29 ± 0.63	0.45 ± 0.28a
B7H1:	1.38 ± 0.71	0.65 ± 0.33a
B7H2:	0.76 ± 0.54	0.38 ± 0.19a
ICOS:	1.62 ± 0.82	0.53 ± 0.27a

^aP<0.05 vs tumor cell.

Figure 1 mRNA expression of B_7-1 (A), B₇H₁ (B), B₇H₂ (C) and ICOS (D) in human colorectal cancer tissues (ISH×400).

Table 2 Relationship between depth of tumor invasion and B7 family molecule expression (mean±SD).

B7 molecule	Tumor cell			TIL
	T2	T3	T4	T2	T3	T4
B7-1:	1.22 ± 0.82	1.28 ± 0.70	1.35 ± 0.42	0.36 ± 0.43	0.50 ± 0.25	0.43 ± 0.24
B7H1:	1.43 ± 0.63	1.55 ± 0.76	1.01 ± 0.63	0.66 ± 0.25	0.76 ± 0.36a	0.42 ± 0.21
B7H2:	0.62 ± 0.05	0.91 ± 0.69	0.56 ± 0.28	0.42 ± 0.21	0.39 ± 0.19	0.33 ± 0.18
ICOS:	1.59 ± 1.14	1.77 ± 0.78	1.35 ± 0.78	0.48 ± 0.35	0.53 ± 0.23	0.59 ± 0.33

^aP<0.05 vs T2, T4.

Table 3 Relation between tumor metastasis and B₇ family molecule expression (mean±SD).

B7 molecule	Tumor cell		TIL
	Metastasis	Nometastasis	Metastasis	Nometastasis
B7-1:	1.08 ± 0.59	1.31 ± 0.64	0.34 ± 0.18	0.46 ± 0.28
B7H1:	0.70 ± 0.00	1.45 ± 0.71a	0.29 ± 0.04	0.68 ± 0.35a
B7H2:	0.59 ± 0.01	0.78 ± 0.57	0.47 ± 0.33	0.37 ± 0.18
ICOS:	0.63 ± 0.04	1.72 ± 0.80a	0.24 ± 0.06	0.56 ± 0.27a

^aP<0.05 vs metastasis.

DISCUSSION

It has been accepted that the efficacy of antitumor immunity is mainly affected by three factors, namely status of tumor immunogenecity, expression of co-stimulatory molecules and type of cytokines. Lack of co -stimulatory molecules in tumor cells might be a reasonable explanation of the tumor immune evasion, but the fact that most human cancer tissues express co-stimulators makes it contradictory. Si et al[3] reported that B_{7 -1} molecule expresses at least in more than half of human solid tumors. MHC-I, II molecules are detectable in human cervical cancer and tumor-associated dendritic cell (DC)[4]. Analysis of tumor microenvironment has demonstrated that type II cytokines are predominant in non-small cell lung cancer and gastric carcinoma[5,6]. Interestingly there is a dynamic change with the progress of tumor, i.e. the expression of IFN-γ and IL-2 decreases, whereas the expression of Th2 cytokines such as IL-4, IL-10 and TNF-β increases significantly.

There is evidence that co-stimulators play a very important role in antigen-specific T cell activation. In vitro and in vivo experiments have shown that T cell interaction via T-cell receptor (TCR) with MHC/antigen complexes in the absence of co-stimulation may lead to induction of anergy rather than activation. B_7-1 gene-modified tumor cells can elicit tumor rejection response in xenograft tumor models. All these suggest that deficiency of co-stimulators is the key factor for immune evasion. However, Dong et al[7] found that CD₂₈ by its natural legend B_{7- 1} or B_7-2 triggers Th1 cytokine secretion including IFN-γ and IL-2, while IL-10 production is increased considerably after stimulation with immobilized B₇H ₁Ig and antibody against CD₃. Dynamic observation has demonstrated that the production of IL-2 peaks 24 h after B₇H₁ co-stimulation, whereas IL-10 secretion starts to increase after 48 and 72 h. Additionally, monoclonal antibody against IL-2 could inhibit IL-10 secretion from T cells co-stimulated with B₇H₁Ig. ICOS, one of the B₇ legends, plays a critical and non-dispensable role in T cell activation and proliferation in vitro, especially in regulating Th2 differentiation. In addition, it is critical for efficient T-cell priming and production of Th2 cytokines in vivo , in particular IL-4 and IL-10. Results obtained from some experiments demonstrated that Ag stimulation of naive CD4⁺ T cells in the absence of ICOS signal results in impaired Th2 development[8]. At the molecular level, ICOS is involved in upregulating the expression of CD40L as well as promoting the secretion of IL-4 and IL-13. ICOS is a co-stimulatory receptor essential for T- cell activation and function and also plays an important role in acquired immunity[9,10]. This could well elucidate our previous findings, i.e. although expression of B_7-1 by human tumor cells can activate T cells and enhance the secretion of Th1 cytokines such as IL-2 and IFN-γ , Th2 cytokine predominates ultimately since co-stimulators such as B₇H₁, B₇H₂ and ICOS act dominantly.

In the present study, we observed that B_7-1 co-stimulatory molecule mRNA was expressed in tumor cells and tumor infiltrating lymphocytes (TIL) of human colorectal carcinoma by in situ hybridization, which is consistent with many experiments of B₇-gene transfection vaccine[11,12]. However, the other members of B₇ family such as B₇H₁, and ICOS can also be detected. In our study, the expression of B₇ molecules in tumor cells was higher than that in TIL (P<0.005), and B₇H₁ and ICOS mRNA expression in tumor cells was even higher than that of B_7-1, suggesting that B₇ family plays a role in tumor immunity. In addition, the expression of B₇H₁ and ICOS mRNA is related with the invasion depth of tumor, the mRNA expression of B₇H₁ and ICOS in both tumor cells and TIL is associated with the metastasis of colorectal carcinoma. A recent study on B₇ family molecules in tumor biological behavior demonstrated that aberrant expression of B₇H₁ in renal cell carcinoma apparently impairs T cell function and survival[13]. Our results and these data provide the morphological and clinical support to elucidate the role of B₇ co-stimulatory molecules in tumor immune evasion.

The present findings indicate that new members of B7 family such as B₇H₁, B₇H₂ and ICOS are involved in promoting TH2- based responses preferentially. More interestingly, it has been reported that tumor-associated B₇H₁ can promote apoptosis of antigen -specific human T-cell clones in vitro[14,15], and mouse P815 tumor -expressed B₇H₁ increases apoptosis of activated tumor-reactive T- cells and promotes the growth of highly immunogenic B tumor in vivo[10]. These results suggest that induction of apoptosis of tumor-infiltrating lymphocytes by B₇H₁ molecules overexpressed in human colorectal carcinoma may be a potentially active escape strategy of various tumors from immune attack as is shown by Wintterle et al[16].

Tumor escape is attributed to a variety of immune evasion strategies, including downregulation of MHC-I class molecules, regulatory T cells, Th2 cytokines, secretion of immunosuppressive factors from tumor cells or TIL, and lack of T-cell co-stimulation. Tumor local microenvironment plays an essential role in determining the final destiny of antitumor immunity[17].

The discovery of new members of B₇ family presenting different co-stimulatory effects indicates that different co-signaling molecules lead to different results, such as enhancement of T-cell Receptor (TCR) -mediated immune responses or inhibition of TCR-mediated immune responses. Tumor -associated B₇H₁ might produce tumor immune escape by promoting apoptosis of tumor-reactive T-cells and type II cytokine secretion through PD-1 ligand[18,19]. Hirano et al[20] reported that B₇H₁ /PD-1 forms a molecular shield to prevent destruction by CTL, suggesting that to block B₇H₁ or PD- 1 by specific monoclonal antibodies could reverse this resistance and profoundly enhance therapeutic efficacy. Strome et al[21] have shown the feasibility of the new immunotherapy. With the suggestion of the concept of co-inhibitors[22], investigation of the functional characteristics of tumor-associated signal molecules may contribute to exploitation of T cell-based tumor immunotherapy.