Fruquintinib Enhances the Antitumor Immune Responses of Anti-Programmed Death Receptor-1 in Colorectal Cancer

Qingli Li, Xiaojiao Cheng, Cong Zhou, Yao Tang, Fuli Li, Baiwen Zhang, Tinglei Huang, Jianzheng Wang, Shuiping Tu, Qingli Li, Xiaojiao Cheng, Cong Zhou, Yao Tang, Fuli Li, Baiwen Zhang, Tinglei Huang, Jianzheng Wang, Shuiping Tu

Abstract

Background: Programmed death receptor-1 (PD-1) blockade shows little benefit in patients with microsatellite-stable colorectal cancer (MSS-CRC). Fruquintinib is a China-made anti-angiogenic drug which is approved for the third line therapy in mCRC. This study investigates the effect of the combination of fruquintinib and PD-1 blockade on MSS-CRC and its relative mechanisms.

Methods: The mouse allograft tumor models that represent MSS and microsatellite instability (MSI) CRC were established using murine CT26 and MC38 colon cancer cells, respectively, to assess the treatment efficacy. The percentages of immune cells were detected in the peripheral blood, spleen and tumor tissues in the tumor-bearing mice by flow cytometry analysis. Angiogenesis in tumor tissues was detected by immunofluorescence. The safety of drug treatment was evaluated by histopathology analysis in murine main organs. The efficacy of the combination of fruquintinib and sintilimab were verified in the treatment of MSS-CRC patients.

Results: Our results showed that the combination of fruquintinib and sintilimab exhibited the strongest inhibition of tumor growth and achieved the longest survival time in mice bearing MC38 or CT26 xenograft tumors, compared to fruquintinib and sintilimab alone. Mechanistically, the combination of fruquintinib and sintilimab reduced angiogenesis, reprogramed the vascular structure, enhanced the infiltration of CD8+T cells (p<0.05), CD8+TNFα+ (p<0.05) T cells and CD8+IFNγ+ (p<0.05) T cells and reduced the ratios of MDSCs and macrophages in mice. There was no obvious toxicity observed in the main organs of the tumor-bearing mice with the combined treatment. Moreover, the treatment using the combination of fruquintinib and sintilimab achieved effective response in five patients with refractory advanced MSS CRC.

Conclusion: Our results show that the combination of fruquintinib and sintilimab greatly inhibits CRC growth by altering tumor immune microenvironment. This study provides the rational for using the combination of fruquintinib and anti-PD-1 antibody for the treatment of advanced CRC.

Keywords: anti-angiogenesis; fruquintinib; immunotherapy; microsatellite stable colorectal cancer; sintilimab.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Li, Cheng, Zhou, Tang, Li, Zhang, Huang, Wang and Tu.

Figures

Figure 1
Figure 1
Fruquintinib inhibits tumor growth in mouse CRC allograft tumor models. Tumor-bearing mice were treated with different doses (2.5, 5, and 10mg/kg) of fruquintinib or CMC-Na control by oral gavage daily for a total of 21days. Tumor volume and immune cells were analyzed on days 14 after fruquintinib treatment (n = 3 per group). (A) The tumor growth curve of MC38 tumor-bearing mice (p = 0.026). (B) the tumor volume measured at the 28th day after treatment (p < 0.0001). (C) MDSCs were analyzed in spleen on day14 after fruquintinib treatment. (D, E) CD8+T cells and CD4+Tcells were analyzed in spleen. (F) MDSCs were analyzed in peripheral blood on day14 after fruquintinib treatment. (G, H) CD8+T cells and CD4+Tcells were analyzed in peripheral blood on day14 after fruquintinib treament. *p < 0.05, ***p < 0.001, ****p < 0.0001. ##p < 0.01,###p < 0.001. (* compared to control group, #compared to Fru2.5 group).
Figure 2
Figure 2
Fruquintinib enhances the anti-tumor effect of anti-PD-1 in CRC mouse models. (A, B) Tumor growth curve of the subcutaneously implanted tumors in MC38 and CT26 mouse models after treatment (n = 4-6). (C, D) Tumor volume of the subcutaneously implanted tumors in each mouse showed in 28 days after treatment (E) Diagram depicting the tumor volume of MC38 allograft tumor model. (F) Tumor weight in the MC38-bearing mice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (G, H) Overall survival of MC38 and CT26 tumor-bearing mice. Kaplan-Meier survival curves were shown in each group of implanted tumors #p < 0.05, compared to Fru2.5 group).
Figure 3
Figure 3
Fruquintinib enhances the effect of sintilimab to reprogram the immune microenvironment and enhance antitumor immunity in CRC. (A) Percentages of CD8+T cells, CD8+TNFα+T cells,CD8+IFNγ+ T cells in tumor tissues determined by FACS in CT26 tumor-bearing mice treated with indicated drugs (shown as fractions of CD45+ cells) in tumors treated with control CMC-Na, anti-PD-1, Fru2.5, arnti-PD-1 and Fru 2.5, measured by flow cytometry at day 14. (B) Representative images of the FACS plots of CD8+ and CD8+IFNγ+ CTLs. (C) IHC images for CD8 staining of tumor sections on day14. Scale bar, 200um. (D) Percentage of MDSCs (CD11b+Gr1+) and TAM (CD11b+F4/80+) in each groups on day 14. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 4
Figure 4
Fruquintinib reduces angiogenesis in CRC both in vitro and in vivo. (A) The effect of fruquintinib on tubular formation in HUVEC in vitro with different doses (0.03,0.3, and 3umol/l) of fruquintinib or CMC-Na control. (B) Representative immunofluorescent staining of sections from different treatment groups on day14. Red, CD31; α-SMA staining; green; blue, DAPI staining. (C) Fruquintinib reduces tumor vascular formation in CT26 colon cancer-bearing mice by angiography using ultrasound machine on day 7 and day 21 after treatment.
Figure 5
Figure 5
A safety of the combination of fruquintinib and anti-PD-1 in vivo. (A) Body weight of tumor-bearing mice in each group. The weight was measured every three days. (B) Blood cell analysis,liver and kidney function and histopathology morphology of the murine major organs (heart, liver, lung, kidney) in the mouse models.
Figure 6
Figure 6
Efficacy of the combination therapy of fruquintinib and anti-PD-1 in patients in MSS mCRC. Radiologic evidence of the 5 patients who achieved PR or SD after the combination treatment. CT scans were performed at the baseline and subsequent treatment cycles. (A) Two CRC patients with lung metastasis: (case 1 # and case 2) were treated with anti-PD-1 and Fruquintinib, and the lesions were significantly reduced (PR). (B) Two CRC patients with liver metastasis: (case 3and case 4) were treated with anti-PD-1 and fruquintinib, the liver metastases showed stable morphological changes, the density became uniform and reduced, the edge became clear, and the peripheral edge enhancement disappeared. (C) One CRC patient with abdominal wall metastasis (case 5) were treated with anti-PD-1 and fruquintinib, and the lesions were significantly reduced (PR). Individual clinical outcomes and the 5 patients enrolled by therapeutic responses (assessed via RECIST v1.1) in Table 1. No, patient number; y, years old; PR, partial response; SD, stable disease; PD, progressive disease.

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