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. 2023 Sep;114(9):3759-3769.
doi: 10.1111/cas.15903. Epub 2023 Jul 13.

Interferon-expressing oncolytic adenovirus + chemoradiation inhibited pancreatic cancer growth in a hamster model

Shuhei Shinoda  1   2 , Nikita S Sharma  1 , Naohiko Nakamura  1 , Kazuho Inoko  1 , Mizuho Sato-Dahlman  1   3 , Paari Murugan  4 , Julia Davydova  1   3 , Masato Yamamoto  1   3   5
Affiliations

Interferon-expressing oncolytic adenovirus + chemoradiation inhibited pancreatic cancer growth in a hamster model

Shuhei Shinoda et al. Cancer Sci. 2023 Sep.

Abstract

Past clinical trials of adjuvant therapy combined with interferon (IFN) alpha, fluorouracil, cisplatin, and radiation improved the 5-year survival rate of pancreatic ductal adenocarcinoma (PDAC). However, these trials also revealed the disadvantages of the systemic toxicity of IFN and insufficient delivery of IFN. To improve efficacy and tolerability, we have developed an oncolytic adenovirus-expressing IFN (IFN-OAd). Here, we evaluated IFN-OAd in combination with chemotherapy (gemcitabine + nab-paclitaxel) + radiation. Combination index (CI) analysis showed that IFN-OAd + chemotherapy + radiation was synergistic (CI <1). Notably, IFN-OAd + chemotherapy + radiation remarkably suppressed tumor growth and induced a higher number of tumor-infiltrating lymphocytes without severe side toxic effects in an immunocompetent and adenovirus replication-permissive hamster PDAC model. This is the first study to report that gemcitabine + nab-paclitaxel, the current first-line chemotherapy for PDAC, did not hamper virus replication in a replication-permissive immunocompetent model. IFN-OAd has the potential to overcome the barriers to clinical application of IFN-based therapy through its tumor-specific expression of IFN, induction of antitumor immunity, and sensitization with chemoradiation. Combining IFN-OAd with gemcitabine + nab-paclitaxel + radiation might be an effective and clinically beneficial treatment for PDAC patients.

Keywords: interferon alpha; nab-PTX; oncolytic adenovirus; oncolytic virus; pancreatic cancer.

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Conflict of interest statement

The authors have no commercial associations that might be a conflict of interest in relation to this article.

Figures

FIGURE 1
FIGURE 1
Viral replication of the interferon (IFN)‐expressing oncolytic adenovirus (IFN‐OAd) combined with chemotherapy. (A) Structure of the IFN alpha‐expressing oncolytic adenovirus (IFN‐OAd). IFN‐OAd is a wild‐type replication oncolytic adenovirus expressing the hamster IFN alpha gene from the adenoviral E3 region. (B–E) Viral replication of IFN‐OAd with gemcitabine (GEM) and paclitaxel (PTX) in vitro. HP‐1 cells were infected with IFN‐OAd. Twenty‐four hours after infection, PBS (Without chemo), GEM (GEM only: 2.5 or 10 nM GEM), PTX (PTX only: 2.5 nM or 10 nM PTX), and GEM+PTX (2.5 nM GEM + 2.5 nM PTX or 10 nM GEM + 10 nM PTX) were added to each well. The viral copy number was measured by quantitative PCR (qPCR) on Day 7. The results are shown as E4 copy number per dish and ratio compared to without chemo (n = 3). Data are presented as mean ± standard deviation (n = 3, *p < 0.05). ADP, adenoviral death protein; Ham‐IFN, hamster interferon alpha; L‐ITR, left inverted terminal repeat; RGD, RGD‐4C (arginine‐glycine‐aspartic) motif; R‐ITR, right inverted terminal repeat.
FIGURE 2
FIGURE 2
Cell viability assay and colony formation assay of interferon (IFN)‐expressing oncolytic adenovirus (IFN‐OAd) combination with chemotherapy and radiation therapy. (A, B) Cell viability assay of IFN‐OAd with chemotherapy. HP‐1 cells were infected with IFN‐OAd (Day 0). Twenty‐four hours after infection, gemcitabine (GEM) and paclitaxel (PTX) were added. On Days 4 and 7, cell viability was assessed (n = 3). (C‐E) Colony formation assay of IFN‐OAd combination with GEM + PTX and radiation therapy. HP‐1 cells were infected with IFN‐OAd (Day 0), and on the following day, cells were irradiated and plated. GEM + PTX was also added. On Day 14, colonies were counted. Data are presented as mean ± standard deviation (n = 3, *p < 0.05). C, chemotherapy; Con, control; R, radiation therapy; V, IFN‐OAd.
FIGURE 3
FIGURE 3
Antitumor effect of interferon (IFN)‐expressing oncolytic adenovirus (IFN‐OAd) combination therapy in hamster syngeneic subcutaneous tumor model. (A) Treatment schedule fot hamsters. HP‐1 cells were subcutaneously inoculated. Once the average tumor volume reached 200 mm3, the hamsters were divided into four groups: Control (n = 4), chemotherapy (gemcitabine [GEM] + nab‐paclitaxel [nab‐PTX]) + radiation (CR) (n = 10), IFN‐OAd (V) (n = 6), and CRV (n = 5) groups. A single dose of virus or vehicle alone (PBS) was injected into the tumors on Day 0. Radiation was performed on Day 3. Intraperitoneal injection of 20 mg/kg GEM + 2 mg/kg nab‐PTX was given twice weekly. (B) The average body weight (g) of each group on Day 21. Data are presented as mean ± standard deviation. (C) Average tumor volume of CRV. The average tumor volumes on Day 21 of the control, CR, V, and CRV were 2027 ± 1296, 908 ± 849, 767 ± 593, and 202 ± 161 mm3, respectively. (D) Individual relative tumor volume from baseline (Day 0). The dashed line represents the baseline (=1). (E–H) Individual tumor volume of control, CR, V, and CRV. (n.s., not significant; *p < 0.05).
FIGURE 4
FIGURE 4
Chemotherapy + radiation + IFN‐OAd‐induced tumor‐infiltrating lymphocytes. (A) Hamster subcutaneous tumors (HP‐1 cells) were excised and processed on Day 21 to assess the tumor‐infiltrating lymphocyte (TIL). Pathologist assessed the TIL in both the intratumoral and the marginal area on hematoxylin and eosin stain sections based on the International Immuno‐Oncology Biomarker Working Group guidelines for TIL assessment in invasive breast carcinoma. (B) After choosing three random areas from both the intratumoral and marginal areas, we scored the average TIL. Necrotic areas were excluded. Data are presented as mean ± standard deviation (n = 3, *p < 0.05, low power field: ×20 magnifications, intratumoral and marginal area: ×200 magnifications). CR, chemotherapy (gemcitabine + nab‐paclitaxel) + radiation; CRV, CR + IFN‐OAd; IFN‐OAd, interferon‐expressing oncolytic adenovirus.
FIGURE 5
FIGURE 5
Chemotherapy + radiation did not hamper virus replication in the tumor. (A) Immunological staining of hamster tumors. Under the same therapies (Figure 3A), the hamsters were killed on Day 7. The expression of adenovirus late gene product (hexon) was assessed by immunostaining with the anti‐hexon polyclonal antibody (counterstained with DAPI). Green: adenovirus hexon protein; blue: nucleus. HE and Masson's trichrome staining showing the similar region of interest (original magnification: ×20) (B) DNA was purified from whole tumors, and the adenoviral DNA copy number of the E4 region was quantified by quantitative PCR (qPCR). The results are shown as E4 copy number per 100 ng DNA. Data are presented as mean ± standard deviation (n = 3; n.s, not significant). DAPI, 4′,6‐diamidino‐2‐phenylindole; HE, hematoxylin and eosin stain; V, IFN‐OAd; CRV, chemotherapy (gemcitabine + nab‐paclitaxel) + V.
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