Chemotherapy agents stimulate cytotoxic T cell against human colon cancer cells through upregulation of the transporter associated with antigen processing

Objectives : Single immunotherapy fails to demonstrate efficacy in patients with microsatellite stable (MSS) metastatic colorectal cancer (mCRC). Research on immune reactions before and after systemic agents for mCRC is warranted. Methods : Our study examined cell line models to compare the expression of immune surface markers on colon cancer cells before and after chemotherapy agents. We also elucidated mechanisms underlying the effects of chemotherapy agents on immune surface markers. We used real-world clinical samples with NanoString analysis and the Perkin-Elmer Opal multiplex system. Results : We established that chemotherapy agents, particularly 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan, stimulated the expression of stimulatory MHC class I isotypes through stimulation the pathway of transporters associated with antigen processing 1 and 2 (TAP1 and TAP2) in cell line models. Application of infected cell protein 47 (ICP-47), a specific inhibitor of the TAP1/TAP2, significantly inhibited expression of TAP1/TAP2 and also inhibited the expression of the downstream MHC class I. We confirmed that the expression of major histocompatibility complex (MHC) class I, programmed death 1(PD-1), and programmed death ligand 1(PD-L1) significantly increased after first-line chemotherapy and targeted therapy in the samples of real-world patients with de novo Conclusion : Our study provides new insights for novel immunotherapy combinations.


Introduction
Colorectal cancer (CRC) is both the third most prevalent cancer worldwide and the third leading cause of cancer death in Taiwan. [1,2] Currently, the treatment backbone of metastatic CRC (mCRC) continues to be chemotherapy and targeted agents. [2][3][4] Immunotherapy with immune checkpoint inhibitors (ICIs), such as anticytotoxic T lymphocyte-associated antigen 4 antibody and anti-programmed-death 1 (PD-1) antibodies, has demonstrated tremendous breakthroughs in treatments for melanoma, renal cell carcinoma, non-small-cell lung cancer, and several other cancer types. [5][6][7][8] By contrast, ICIs are applied only for patients with microsatellite instability high (MSI-H) mCRC. [9,10] However, MSI-H mCRC accounts for only 1.8%-4.0% of all patients with mCRC. [11] A possible immune pathway must be surveyed to develop a new strategy for immunotherapy in mCRC treatment.
The immune microenvironment exerts effects on the efficacy and delivery of chemotherapy and targeted therapy. [12,13] Chemotherapy and targeted therapy might change the tumor microenvironment and immune surface markers. [14][15][16] Irinotecan increases the endoplasmic reticulum stress of tumor cells, subsequently inducing immunogenic cell death (ICD). [17] Oxaliplatin inhibits pSTAT6 in tumor cells, which subsequently inhibits the expression of PD-L2 and increases the expression of major histocompatibility complex (MHC) class I, namely human leukocyte antigen (HLA) class I. Both actions enhance the efficacy of cytotoxic T cells. [18,19] Moreover, oxaliplatin induces the expression of calreticulin in tumor cells and thus enhances ICD. [16,19]. Fluorouacil (5-FU) stimulates the cytotoxicity of natural killer (NK) cell and also stimulates the expression of MHC class I. [20,21]. Cetuximab, an antiepidermal growth factor receptor monoclonal antibody, can stimulate immune effector cells and induce antibody-dependent cell-mediated cytotoxicity (ADCC). [22,23] Bevacizumab, an antivascular endothelial growth factor monoclonal antibody, targets endothelial cells in peritumor parts and exerts immune modulating effects by influencing the tumor microenvironment. [24,25] The aforementioned agents are all standard systemic agents for the treatment of mCRC. That is, research on immune reactions before and after systemic agents for mCRC is warranted for new immunotherapy targets.
Our study plans to incorporate data from real-world clinical samples into cell line models. We compared the expression of immune surface markers in colon cancer cells before and after chemotherapy agents and elucidated mechanisms underlying the effects of chemotherapy agents on immune surface markers. We would focus on the interactions between chemotherapy agents and antigen processing pathway and the subsequent dynamic change of MHC class I. [26][27][28] And we also applied interferon-γ as positive control [29,30].

IFN-γ specifically stimulated the expression of stimulatory MHC class I isotypes.
First, we tested three colon cancer cell lines, SW480, COLO 320 and HT29, through flow cytometry. These cell lines all initially exhibited low expression of MHC class I and NK cell ligands (Fig. 1A). The expression of MHC class I significantly increased after IFN-γ stimulation. By contrast, NK cell ligands including MIC A/B and ULBP-1 were both irresponsive to IFN-γ. We then tested more isotype expressions specifically on SW480. SW480 demonstrated low expression of all MHC class I isotypes, including pan-MHC class I, HLA-A, HLA-C, HLA-E, HLA-F, and HLA-G. NK cell ligands, including MIC A/B and ULBP, both exhibited low expression for SW480 cells. IFN-γ specifically stimulated the expression of MHC class I, particularly HLA-A, but NK cell ligands were responsive to IFN-γ stimulation. In summary, IFN-γ significantly stimulates the expression of MHC class I, particularly HLA-A without stimulating NK cell ligands (Fig. 1B). Although it was non-significant, a mild trend occurred whereby the stimulatory effect of MHC class I and HLA-A in response to IFN-γ stimulation was positively correlated with IFN-γ dosage and incubation time.

Discussion
In our study, we indicated that the expression of MHC class I, PD-1, and PD-L1 Our study might illuminate the subject of immunotherapy in mCRC treatment and provide new rationale for novel immunotherapy combinations. Chemotherapy agents were the backbone of treatment in the targeted therapy era and continue to be so in the immunotherapy era.

Cell lines
We used a panel of colon cancer cell lines, including SW480, HT29, and COLO320. All cell lines were purchased from the American Type Culture Collection.

Chemicals and other reagents
Compounds used in this study for colon cancer cell lines were 7-ethyl-10-

Flow cytometry
We seeded 10 6 cells with 10-mL culture medium in 10-cm dishes. The next day, MA, U.S.) and exposed to film for 1 to 10 minutes.

Protein Transfection
We first seeded 4 × 10 5 cells with 3 mL of culture on a 6-well plate. The next day, TAP-inhibitor ICP47 (5 μg) was added to the tube containing the Xfect protein transfection reagent and incubated at room temperature for 30 minutes. The mixture was then applied to cells with 400 μL of serum-free medium (RPMI-1640 only) in each well and incubated at 37°C for 60 minutes. We added 3 mL of the culture medium to each well and performed incubation at 37°C for another 2 hours. SN-38 (0.1 μM) was treated for 24 or 48 hours. As a control, some other cells were stained with X-gal to determine the transfection efficiency through beta-galactosidase control (Takara Bio, 631326, Mountain View, CA, U.S.)

Isolation of PBMCs
PBMCs were isolated from peripheral blood of the health human volunteers. We applied Ficoll-Paque Plus and diluted with PBS by density gradient centrifugation (400 g, 20 minutes, without break). Then, the monocytes were purified by positive selection with human CD14 + magnetic MicroBeads by manual MACS cell separation system (Miltenyi Biotec). The monocytes were incubated for 6 days into RPMI-1640 and supplemented with 10% FBS, 1% Antibiotic-Antimycotic, 50 ng/mL GM-CSF and 20ng/mL IL-4 in an atmosphere of 95% O2 and 5% CO2 at 37°C to harvest MoDCs.
The induction medium would be renewed at the third day.

Patient enrollment
We enrolled one patient who had been treated at National Taiwan

Immunohistochemical (IHC) staining of patient samples
We applied the Perkin-Elmer Opal multiplex system to simultaneously detect multiple biomarkers plus nuclear counterstain within a single image. Initially, the 5-μm FFPE pathology slides were incubated at 70°C for 1.5 hours, deparaffinized with xylene, and then hydrated through an ethanol gradient ending with distilled water wash. The slides were fixed using 10% neutral buffered formalin for 20 minutes. Antigen

Statistical analysis
All results were collected with at least three or more independent tests. The testing results were presented in average and standard deviations were also demonstrated within figures. We also applied the ImageJ for quantification of western